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REVIEW
Optical techniques for the noninvasive diagnosis of skin cancer
Mihaela Antonina Calin • Sorin Viorel Parasca •
Roxana Savastru • Marian Romeo Calin •
Simona Dontu
Received: 4 March 2013 / Accepted: 21 March 2013 / Published online: 4 April 2013
� Springer-Verlag Berlin Heidelberg 2013
Abstract
Purpose The aim of this paper is to provide an overview
of the most investigated optical diagnostic techniques:
optical coherence tomography, fluorescence spectrometry,
reflectance spectrometry, Raman spectroscopy, and con-
focal microscopy.
Methods A search of three databases was conducted
using specific keywords and explicit inclusion and exclu-
sion criteria for the analysis of the performances of these
techniques in the pre- and postoperative diagnosis of skin
cancers.
Results Optical coherence tomography has shown prom-
ising results in the assessment of deep margins of skin
tumors and inflammatory skin diseases, but differentiating
premalignant from malignant lesions proved to be less
effective. Fluorescence spectroscopy proved to be effective
in revealing the biochemical composition of tissue; early
detection of malignant melanoma was reliable only with
stepwise two-photon excitation of melanin, while tumoral
margin assessment and differential diagnosis between
malignant and non-malignant lesions showed some con-
flicting results. Characterization of the structural properties
of tissue can be made using diffuse reflectance spectrom-
etry, and the values of the specificity and sensitivity of this
method are ranging between 72–92 % and 64–92 %,
respectively. Raman spectroscopy proved to have better
results both in carcinoma and melanoma diagnosis with
sensitivities and specificities above 90 % and high above
50 %, respectively. Confocal microscopy is the closest
technique to pathological examination and has gained the
most clinical acceptance, despite the need for a standardi-
zation of the interpretation algorithm.
Conclusions In conclusion, these optical techniques
proved to be effective in the diagnosis of skin cancer, but
further studies are needed in finding the appropriate
method or combination of methods that can have wide
clinical applications.
Keywords Optical coherence tomography � Fluorescence
spectrometry � Reflectance spectrometry � Raman
spectroscopy � Confocal microscopy � Sensitivity and
specificity indices
Introduction
The need for more objective and quantitative methods to
support the diagnosis is a priority for physicians, biologists,
physicists, and engineers, and new optical imaging and
spectroscopic techniques have been developed in order to
answer to this demand. Optical techniques can provide
M. A. Calin (&) � R. Savastru � S. Dontu
National Institute of Research and Development for
Optoelectronics INOE 2000, 409 Atomistilor Street,
P.O. Box MG5, 077125 Magurele, Ilfov, Romania
e-mail: [email protected]; [email protected]
R. Savastru
e-mail: [email protected]
S. Dontu
e-mail: [email protected]
S. V. Parasca
Emergency Clinical Hospital for Plastic, Reconstructive Surgery
and Burns, 218 Grivitei Street, Bucharest, Romania
e-mail: [email protected]
M. R. Calin
‘‘Horia Hulubei’’ National Institute for Physics and Nuclear
Engineering, IFIN HH, 30 Reactorului Street, Magurele,
Ilfov, Romania
e-mail: [email protected]
123
J Cancer Res Clin Oncol (2013) 139:1083–1104
DOI 10.1007/s00432-013-1423-3
noninvasive, low-cost methods for a variety of applica-
tions. Techniques like optical coherence tomography
(OCT) and fluorescence imaging have been widely evalu-
ated for imaging of the retina and cancer diagnosis,
respectively (Framme et al. 2005; Drexler and Fujimoto
2008; He et al. 2010; Alex et al. 2011). Reflectance spec-
troscopy is a well-known method in getting information on
optical properties of tissue that can be used in skin cancer
evaluation (Morales and Montiel 2012). Raman spectros-
copy (RS) and confocal microscopy have gained some
clinical acceptance after multiple trials (de Paula and Sa-
thaiah 2005; Amjadi et al. 2011). All these methods offer,
with minimal costs, new ways of accurate differential
diagnosis between benign and malignant skin lesions.
This article aims to present five of all these techniques
used for the diagnosis of skin cancer (OCT, fluorescence
spectrometry, reflectance spectrometry, Raman spectros-
copy, and confocal microscopy) and to analyze their value,
based on a review of the available literature. Several other
promising optical techniques such as near-infrared spec-
troscopy, differential pathlength spectroscopy, and coher-
ent backscattering spectroscopy are not included in this
study because they are relatively less studied in clinical
diagnosis of skin diseases.
This study will try to answer the following questions: is
there a place for clinical use of these methods in skin
cancer evaluation? What devices/parameters can be used in
optical diagnostics? What is the specificity and selectivity
of optical methods for diagnosis of skin malignancies?
Methods
A review of the literature published between 2002 and
2012 on the optical methods for the diagnosis of skin
cancers was performed. A rigorous search of major dat-
abases (including MEDLINE, CANCERLIT, and PubMed)
was performed using specific keywords. ‘‘optical diagno-
sis’’ was associated with: ‘‘skin,’’ ‘‘cancer,’’ ‘‘cancerous,’’
‘‘malignant,’’ ‘‘precancerous,’’ ‘‘premalignant,’’ ‘‘derma-
tology,’’ and the following phrases used as text words:
‘‘skin cancer,’’ ‘‘benign lesions,’’ ‘‘skin imaging,’’ ‘‘clinical
diagnosis of skin disease,’’ ‘‘spectroscopic method,’’
‘‘imaging method,’’ ‘‘optical coherence tomography,’’
‘‘OCT,’’ ‘‘fluorescence spectrometry,’’ ‘‘reflectance spec-
trometry,’’ ‘‘Raman spectrometry,’’ ‘‘confocal micros-
copy,’’ ‘‘sensitivity and specificity of diagnostic.’’
The relevant articles and abstracts that met the above
criteria were selected for inclusion: (a) reviews, guidelines,
and clinical trials, and (b) the device type, parameters, and
sensitivity and specificity indices of the method were
reported. We have excluded articles that dealt with labo-
ratory experiments, although some of them were the bases
for clinical trials included in our research. The papers
published in a language other than English were also
excluded.
Results
Optical coherence tomography (OCT)
Advances in optics, fiber, and laser technology have
enabled the development of a novel noninvasive optical
biomedical imaging technique, OCT. The first in vitro
tomogram of the human eye was presented in 1991. In
dermatology, OCT was introduced in 1995 (Schmitt et al.
1995) and is now increasingly used in clinical skin research
(Gambichler et al. 2005).
Optical coherence tomography is an interferometric,
noninvasive tomographic imaging technique that generates
cross-sectional 2D and 3D images of backscattered or
back-reflected light from the tissue, ideal for real-time
clinical applications such as in vivo surgical monitoring.
OCT represents one of the best options, in terms of depth of
skin penetration and resolution, for collecting morpholog-
ical data of the skin. In its basic configuration, OCT sys-
tems employ Michelson interferometer. The light is split
into two arms: a sample arm and a reference arm. An
interference pattern is obtained only when the length of the
reference arm of interferometer corresponds to the length
of the sample arm within the coherence length of the light
source.
Optical coherence tomography has been used until now
as high-resolution imaging technique in the diagnosis of
various diseases of: eyes (Bijlsma and Stilma 2005; Fig-
urska et al. 2010), gastrointestinal tract (Sivak et al. 2000;
Chen et al. 2007), vascular tissue (Farooq et al. 2009),
dental tissue (Fried et al. 2002; Otis et al. 2003), and skin.
The utility and the accuracy of OCT technique in diag-
nosing and monitoring skin cancerous and non-cancerous
tumors are presented in Table 1.
We can see in Table 1 that the most clinical studies of
the OCT have focused on basal cell carcinoma (BCC).
Studies performed by Jorgensen et al. (2008) and Mogen-
sen et al. (2009), on approximately 130 patients with 200
BCC lesions, have indicated that OCT images of BCC
exhibit characteristic dark circular structures as compared
with normal skin that has a layered structure. Some chan-
ges in the epidermal architecture and flattening the upper
layer of dermis can also be observed. Analysis of these key
indicators that fit well with histopathological test suggest
that OCT can be used in the diagnosis of BCC. The main
limitation of OCT in diagnosing BCC is its inability to
identify subtypes of BCC (Mogensen et al. 2009).
Assessment of the deep margins of the BCC is very
1084 J Cancer Res Clin Oncol (2013) 139:1083–1104
123
Ta
ble
1A
pp
lica
tio
no
fO
CT
tech
niq
ue
for
the
dia
gn
osi
so
fsk
inca
nce
r
Dis
ease
Dev
ice/
par
amet
ers
Res
ult
sR
efer
ence
Bas
alce
llca
rcin
om
a
(BC
C)
M2
-OC
Tsy
stem
(Lig
htL
abIm
agin
gIn
c.,
Wes
tfo
rd,
MA
,U
SA
)
Tw
enty
pat
ien
tsw
ith
bio
psy
-co
nfi
rmed
BC
Cw
ere
dia
gn
ose
db
yO
CT
.D
epth
of
the
neo
pla
smw
asd
eter
min
edth
rou
gh
aco
mp
ute
r-g
ener
ated
dep
thsc
ale
and
dir
ect
mea
sure
men
to
nan
alo
go
us
tiss
ue
spec
imen
su
sin
ga
mic
rosc
op
em
icro
met
er.
Th
e
resu
lts
hav
ed
emo
nst
rate
da
go
od
corr
elat
ion
bet
wee
nti
ssu
eth
ick
nes
sd
eter
min
ed
by
OC
Tan
dro
uti
ne
his
tolo
gic
test
s
Olm
edo
etal
.(2
00
7)
Bas
alce
llca
rcin
om
a–
Tw
enty
-th
ree
pat
ien
tsw
ith
49
lesi
on
sw
ere
sele
cted
for
the
stu
dy
.B
asal
cell
carc
ino
ma
was
iden
tifi
edin
20
pat
ien
ts.
Th
ere
sult
sd
emo
nst
rate
dth
atth
ere
is
exce
llen
tco
rrel
atio
nb
etw
een
OC
Tim
ages
and
his
top
ath
olo
gic
feat
ure
so
f
sup
erfi
cial
,n
od
ula
r,m
icro
no
du
lar,
and
infi
ltra
tiv
eb
asal
cell
carc
ino
mas
.T
he
pre
dic
tiv
ev
alu
eo
fO
CT
has
no
tb
een
eval
uat
edd
ue
toth
esm
all
nu
mb
ero
fp
atie
nts
Olm
edo
etal
.(2
00
6)
Bas
alce
llca
rcin
om
aS
wep
t-S
ou
rce-
OC
T-S
yst
em(O
CS
13
00
SS
,
Th
orl
abs,
Dac
hau
/Mu
nic
h)
k=
1,3
25
nm
Ban
dw
idth
=1
00
nm
Ax
ial
reso
luti
on
=1
2l
m
Lat
eral
reso
luti
on
=1
5lm
Ten
BC
Cs
fro
m1
0p
atie
nts
wer
ein
clu
ded
inth
isst
ud
y.
Th
eac
cura
cyo
fO
CT
and
hig
h-f
req
uen
cyu
ltra
sou
nd
(HF
US
)m
easu
rem
ents
inv
ivo
was
com
par
ed.
Th
e
resu
lts
dem
on
stra
ted
that
OC
Tw
assu
per
ior
toH
FU
Sin
term
so
ftu
mo
rth
ick
nes
s
asse
ssm
ent
Hin
zet
al.
(20
11
)
Bas
alce
llca
rcin
om
a–
Inth
isst
ud
y,
22
BC
Cs
wer
eex
amin
edb
yn
ewh
igh
-defi
nit
ion
OC
T(H
D-O
CT
)u
sin
g
the
en-f
ace
and
slic
eim
agin
gm
od
e.T
he
char
acte
rist
icm
orp
ho
log
icfe
atu
res
of
BC
Cw
ere
eval
uat
edin
com
par
iso
nw
ith
the
his
top
ath
olo
gic
alte
stre
sult
s.R
esu
lts
ob
tain
edin
this
stu
dy
hav
esh
ow
nth
atin
the
en-f
ace
mo
de,
the
lob
ula
ted
stru
ctu
re
of
the
BC
Cw
asm
ore
dis
tin
ctth
anin
the
slic
em
od
eco
mp
ared
toh
isto
log
y.
Th
ese
resu
lts
dem
on
stra
teth
atH
D-O
CT
isb
ette
rth
anco
nv
enti
on
alO
CT
giv
ing
add
itio
nal
info
rmat
ion
inth
ed
iag
no
sis
of
BC
C
Mai
eret
al.
(20
12
)
Bas
alce
llca
rcin
om
a
(BC
C)
acti
nic
ker
ato
sis
(AK
)
OC
Tsc
ann
er(R
iso
eN
atio
nal
Lab
ora
tory
,
Ro
skil
de,
Den
mar
k)
k=
1,3
10
nm
Ban
dw
idth
=6
0n
m
P=
20
mW
Ax
ial
reso
luti
on
=1
0m
m
Lat
eral
reso
luti
on
=2
0m
m
Fo
rty
-on
eB
CC
and
37
AK
lesi
on
sfr
om
34
pat
ien
tsw
ere
enro
lled
inth
isst
ud
y.
Th
e
lesi
on
sw
ere
loca
ted
ind
iffe
ren
tar
eas
of
the
bo
dy
:sc
alp
,fa
ce,n
eck
,lo
wer
bac
kan
d
up
per
extr
emit
ies.
Th
ere
sult
sd
emo
nst
rate
dth
atO
CT
imag
eso
fB
CC
sex
hib
itd
ark
glo
bu
les
corr
esp
on
din
gto
bas
alo
idis
lan
ds.
Fo
rA
Kle
sio
ns,
wh
ite
do
tsan
dst
reak
s
corr
esp
on
din
gto
hy
per
ker
ato
sis
wer
eid
enti
fied
inO
CT
imag
es.
Cla
ssifi
cati
on
accu
raci
eso
f7
3%
(AK
)an
d8
1%
(BC
C)
hav
eb
een
ach
iev
edu
sin
gm
ach
ine-
lear
nin
gan
aly
sis
Jorg
ense
net
al.
(20
08
)
Bas
alce
llca
rcin
om
a
acti
nic
ker
ato
sis
(AK
)
ben
ign
lesi
on
s
OC
Tsy
stem
(Tec
hn
ical
Un
iver
sity
of
Den
mar
k)
k=
1,3
18
nm
Ban
dw
idth
=6
6n
m
Ax
ial
reso
luti
on
=8
lm
Lat
eral
reso
luti
on
=2
4lm
On
eh
un
dre
dfo
ur
pat
ien
tsw
ith
17
6le
sio
ns
wer
ere
cru
ited
.S
ensi
tiv
ity
was
79
–9
4%
and
spec
ifici
tyw
as8
5–
96
%in
dif
fere
nti
atin
gn
orm
alsk
infr
om
lesi
on
s.L
oca
tio
n
of
bas
alce
llca
rcin
om
aw
asin
areo
lam
amm
ae
Mo
gen
sen
etal
.(2
00
9)
J Cancer Res Clin Oncol (2013) 139:1083–1104 1085
123
important in orienting surgical treatment, and OCT proved
to have a good correlation with pathological examination
(Olmedo et al. 2006, 2007; Hinz et al. 2011) and was
superior to ultrasound imaging (Hinz et al. 2011).
A number of studies brought evidence of the perfor-
mance of OCT technique in actinic keratosis (AK) diag-
nosis (Barton et al. 2003; Korde et al. 2007; Jorgensen
et al. 2008; Mogensen et al. 2009). These studies indicate
that AK, characterized by dysplasia and architectural dis-
order of the epidermis, appears in OCT images as white
dots and streaks. The OCT accuracy in the diagnosis of AK
has been assessed only in two studies (Korde et al. 2007;
Jorgensen et al. 2008). In distinguishing AK from normal
skin, a sensitivity of 79–86 % and a specificity of
83–100 % were found for OCT technique. It proved dif-
ficult to distinguish between AK and BCC using OCT.
However, Jorgensen et al. (2008) using machine-learning
analysis have developed classification models able to dif-
ferentiate AK from BCC with a success rate of 73 % and
BCC from AK with a success rate of 81 %.
In parallel with the development of medical applications
of OCT, new OCT systems for dermatological purposes
have been developed, some of which are already com-
mercially available (e.g., LightLab Imaging Inc., Westford,
MA, USA; OCS1300SS, Thorlabs, Dachau/Munich;
BBS1310, JDS Uniphase, Ontario, Canada). Recent pro-
gress in the field of optoelectronics has led to more effi-
cient OCT systems such as high-definition OCT (HD-
OCT) system. The use of such OCT systems in derma-
tology may increase the diagnostic accuracy (Maier et al.
2012).
Finally, we can conclude that OCT can assess deep
limits of soft tissue lesions with an axial resolution of
3–15 lm, which proves to be an advantage in evaluating
certain lesions. As a disadvantage, OCT cannot offer bio-
chemical information. This kind of data can be obtained
with noninvasive and sensitive fluorescence techniques or
diffuse reflectance spectrometry.
Fluorescence techniques
Fluorescence techniques are currently used in clinical
practice for both noninvasive diagnosing and monitoring of
the medical treatment. The principle of fluorescence
diagnosis is based on the interaction between light with a
specific wavelength (usually from UV–Vis range) and
endogenous or exogenous fluorophores present in biolog-
ical tissue. The fluorescence phenomenon has been
described accurately by physicists in the first half of the
twentieth century. Briefly, when a molecule (fluorophore)
absorbs energy in the form of photons of visible/ultraviolet
light, it passes from its ground electronic state to one of the
various vibrational states in the higher-energy excitedTa
ble
1co
nti
nu
ed
Dis
ease
Dev
ice/
par
amet
ers
Res
ult
sR
efer
ence
Su
n-d
amag
esk
inac
tin
ic
ker
ato
sis
(AK
)
BB
S1
31
0JD
SU
nip
has
e(O
nta
rio
,C
anad
a)
k=
1,3
10
nm
Ban
dw
idth
=5
0n
m
P=
11
.5m
W
Ax
ial
reso
luti
on
=1
2l
m
Lat
eral
reso
luti
on
=1
2lm
OC
Tim
ages
wer
eac
qu
ired
of
11
2p
atie
nts
,w
ith
asu
bse
qu
ent
bio
psy
.O
CT
imag
e
anal
ysi
sre
vea
led
ast
atis
tica
lly
sig
nifi
can
td
iffe
ren
ce(P
\0
.00
01
)b
etw
een
the
aver
age
atte
nu
atio
nv
alu
eso
fsk
inw
ith
min
imal
and
sev
ere
sola
rel
asto
sis
and
bet
wee
nu
nd
isea
sed
skin
and
AK
.T
he
resu
lts
of
this
stu
dy
also
dem
on
stra
ted
that
AK
cou
ldb
ed
isti
ng
uis
hed
fro
mu
nd
isea
sed
skin
wit
h8
6%
sen
siti
vit
yan
d8
3%
spec
ifici
ty
Ko
rde
etal
.(2
00
7)
Su
n-d
amag
edsk
inac
tin
ic
ker
ato
sis
BB
S1
31
0,
JDS
Un
iph
ase
(On
tari
o,
Can
ada)
k=
1,3
10
nm
Ban
dw
idth
=5
0n
m
P=
11
.5m
W
Ax
ial
reso
luti
on
=1
2l
m
Inth
isst
ud
y2
0p
atie
nts
wit
hsu
n-d
amag
edsk
inan
dac
tin
ick
erat
ose
sw
ere
inv
esti
gat
ed.
OC
Tim
age
anal
ysi
sre
vea
led
that
:
Su
n-d
amag
edsk
inis
char
acte
rize
db
yin
crea
sed
sig
nal
inth
eep
ider
mis
and
rap
id
atte
nu
atio
no
fli
gh
t;
Act
inic
ker
ato
sis
isch
arac
teri
zed
by
hig
hsu
rfac
ere
flec
tio
n,
the
pre
sen
ceo
fa
low
-
sig
nal
ban
din
the
stra
tum
corn
eum
,an
dh
eter
og
eneo
us
app
eara
nce
inth
e
epid
erm
is/d
erm
is;
OC
Tm
eth
od
pre
sen
tsa
sen
siti
vit
yo
f7
9%
and
spec
ifici
ty1
00
%fo
rA
K
Bar
ton
etal
.(2
00
3)
1086 J Cancer Res Clin Oncol (2013) 139:1083–1104
123
electronic state. From this excited state, the molecule may
return to the basic state by radiative (fluorescence and
phosphorescence) or non-radiative processes (internal
conversion, non-radiation relaxation, and intersystem
crossing). The fluorescence emission appears when the
molecule in excited state loses energy gained by the
absorption of the photon initially before return to lower
energy states. This means that the emitted fluorescence has
a lower energy than the absorbed light, so the wavelength
of emitted fluorescence radiation is higher than that of the
light absorbed since the photon energy is inversely pro-
portional with its wavelength. Consequently, the fluores-
cence spectrum of a molecule will be shifted to higher
wavelengths in comparison with the absorption spectrum.
The intensity of radiation emitted by fluorescence is pro-
portional to that of incident radiation. This is understand-
able because the number of molecules excited by the
absorption of photons is proportional to the intensity of the
incident radiation. Therefore, in order to obtain intense
fluorescence radiation, the sample must be exposed to
intense light radiation fluxes using high-intensity radiation
sources and/or focusing radiation on the sample to be
analyzed. As light sources, both incoherent (lamps, LED)
and coherent light sources (lasers) can be used if they emit
radiation that can be absorbed by the fluorophores present
in the investigated sample.
Human skin contains several native fluorophores such as
NADH, collagen, elastin, tryptophan, tyrosine, and por-
phyrins. (Richards-Kortum and Sevick-Muraca 1996) The
fluorescent properties of these endogenous fluorophores
have been studied widely until now, in correlation with the
alterations in biochemical composition and tissue archi-
tecture induced by different pathologies. These studies
have demonstrated that fluorescence technique [known as
autofluorescence technique (AFT)] is a valuable tool for
in vivo detection of various skin diseases and pathologies,
especially skin cancer. The AFT has some advantages and
disadvantages. The main advantages are its noninvasive-
ness, the real-time response, and low cost (it is not nec-
essary to administer a drug to detect tissue fluorescence).
The disadvantages of AFT are related to: low intensity of
fluorescence radiation emitted by endogenous fluorophores,
difficulty to distinguish among different fluorophores
(because their emission and excitation spectral bands are
wide enough so that often appears a spectral overlapping),
and the complexity of interactions among different
endogenous fluorochromes.
Tissue fluorescence can be improved by the adminis-
tration of exogenous fluorophores (photosensitizers) with
specific absorption and fluorescence properties which
preferentially accumulate in diseased cells (especially
cancer cells) and which, under the action of light radiation
with specific wavelength, emit characteristic fluorescence
radiation highlighting the injurious area. Such a technique
is known as drug-induced fluorescence technique (DFT).
The development of DFT for tissue diagnostics has come
from parallel developments in photodynamic therapy of
malignant lesions with fluorescent photosensitizers.
Clinical studies have demonstrated that DFT has a sig-
nificant diagnostic advantage over AFT due to the increase
in the fluorescence intensity by using exogenous fluoro-
phores, but rises some problems related to: the choice of
photosensitizer type, time interval between the photosen-
sitizer administration and its exposure to light (which
increases response time), side effects induced by photo-
sensitizer administration, costs, and of course, necessary
regulatory approvals.
Detection of fluorescence radiation (either autofluores-
cence or drug-induced fluorescence) may be achieved by
two distinct ways: fluorescence spectroscopy and fluores-
cence imaging. Both techniques have become important
modalities of investigation in clinical practice particularly
in identification and localization of pre- and early cancer-
ous lesions (Table 2).
We can notice from Table 2 that most of the clinical
dermatology research on fluorescence diagnosis has
focused, over the past decade, on the detection or diagnosis
of melanoma and non-melanoma skin cancer as well as in
demarcation of various skin cancers. Studies on early
melanoma detection show that only the stepwise two-
photon excitation of melanin autofluorescence gave some
encouraging results in differential diagnosis between
benign and malignant pigmented lesions (due to a low
melanin autofluorescence) (Eichhorn et al. 2009; Leupold
et al. 2011), but fluorescence was not compared with other
noninvasive methods (like dermoscopy). Photobleaching
analysis seems to generate some specific patterns, but the
study takes into account many different lesions (Lihachev
et al. 2011), while NIR fluorescence gives a different pat-
tern for many lesions compared to normal skin, but does
not show much clinical interest in terms of differential
diagnosis (Huang et al. 2006).
Studies trying to assess the effectiveness of DFT in
determining the BCC’s lateral tumoral margins showed
mostly discouraging results (Gambichler et al. 2008;
Wetzig et al. 2010; Kamrava et al. 2012), the method is
being less accurate than clinical examination. MAL-
induced PpIX fluorescence imaging using fluorescence
image analysis showed better results (Neus et al. 2008), but
further studies are needed before the method becomes
applicable. Early detection of skin malignancy (non-mel-
anoma) was also a subject of investigation, DFT being
evaluated mostly in comparison with histological exami-
nation. The results are conflicting from article to article,
some of them showing good results in both sensibility and
sensitivity of the method (Neus et al. 2008; Kamrava et al.
J Cancer Res Clin Oncol (2013) 139:1083–1104 1087
123
Ta
ble
2A
pp
lica
tio
ns
of
flu
ore
scen
cete
chn
iqu
esfo
rth
ed
iag
no
sis
of
skin
can
cer
Dis
ease
Flu
ore
scen
ce
tech
niq
ue
Dev
ice/
par
amet
ers
Res
ult
sR
efer
ence
Mel
ano
ma
AF
TN
ano
seco
nd
lase
rp
uls
es
k excit
ati
on
=8
10
nm
On
the
bas
iso
fa
new
lyd
evel
op
edm
eth
od
tose
lect
ivel
yex
cite
mel
anin
flu
ore
scen
ceo
fsk
inti
ssu
eb
yst
epw
ise
two
-ph
oto
nex
cita
tio
n,
the
auth
ors
of
this
stu
dy
inv
esti
gat
edin
form
atio
nfr
om
this
mel
anin
flu
ore
scen
cew
ith
resp
ect
toth
ed
iffe
ren
tiat
ion
of
pig
men
ted
lesi
on
s.T
he
resu
lts
rev
eale
da
dis
tin
ctd
iffe
ren
ceb
etw
een
the
mel
anin
flu
ore
scen
ce
spec
tru
mo
fm
alig
nan
tm
elan
om
a(i
ncl
ud
ing
mel
ano
ma
insi
tu)
and
flu
ore
scen
cesp
ectr
um
of
ben
ign
mel
ano
cyti
cle
sio
ns
(i.e
.,co
mm
on
nev
i)
for
fres
hly
exci
sed
sam
ple
sas
wel
las
for
his
top
ath
olo
gic
alsa
mp
les.
A
spec
ific
flu
ore
scen
cew
asal
sore
cord
edfo
rd
ysp
last
icn
evi.
Th
ese
resu
lts
pro
ve
that
earl
yd
etec
tio
no
fm
alig
nan
tm
elan
om
aca
nb
eac
hie
ved
by
AF
T
Eic
hh
orn
etal
.(2
00
9)
Mel
ano
cyti
cn
evi
mal
ign
ant
pig
men
ted
mel
ano
ma
AF
T–
Inth
isst
ud
y,
usi
ng
an
ewm
od
eo
fst
epw
ise
two
-ph
oto
nex
cita
tio
n,
mel
anin
-do
min
ated
flu
ore
scen
cesp
ectr
ao
fp
igm
ente
dsk
inle
sio
ns
are
rep
ort
ed.
Th
ere
sult
so
fth
isst
ud
yre
vea
led
that
the
pu
rem
elan
in
flu
ore
scen
cesp
ectr
ao
fn
orm
alp
igm
ente
dsk
in,
mel
ano
cyti
cn
evi,
and
mal
ign
ant
pig
men
ted
mel
ano
ma
sho
wd
isti
nct
lyd
iffe
ren
tsp
ectr
al
shap
es.
Mel
ano
ma
gav
ea
char
acte
rist
icfi
ng
erp
rin
tw
ith
afl
uo
resc
ence
ban
dp
eak
ing
at6
40
nm
,in
dep
end
ent
of
the
mel
ano
ma
sub
typ
e.T
he
mel
anin
flu
ore
scen
cesp
ectr
ap
eak
edat
59
0n
mfo
ral
lty
pes
of
com
mo
n
mel
ano
cyti
cn
evi.
Ina
seri
eso
f1
67
case
sw
ith
mel
ano
cyti
cn
evi
and
mel
ano
mas
,th
ese
nsi
tiv
ity
of
this
new
met
ho
dto
dia
gn
ose
mel
ano
ma
was
93
.5%
,th
esp
ecifi
city
80
.0%
,an
dth
ed
iag
no
stic
accu
racy
82
.6%
Leu
po
ldet
al.
(20
11
)
Pig
men
ted
and
vas
cula
rle
sio
ns
AF
TT
he
flu
ore
scen
ceex
per
imen
tal
setu
p:
cwla
ser,
op
tica
lfi
ber
bu
nd
lean
d
Av
aSp
ec-2
04
8-2
spec
tro
met
er
k excit
ati
on
=5
32
nm
DP
=6
5m
W/c
m2
Inth
isst
ud
y,
14
1p
igm
ente
dan
dv
ascu
lar
lesi
on
sw
ere
inv
esti
gat
edb
y
lase
r-in
du
ced
skin
auto
flu
ore
scen
cep
ho
tob
leac
hin
gan
aly
sis.
Th
ere
sult
s
of
this
stu
dy
rev
eale
dth
at:
each
of
skin
pat
ho
log
ies
has
asp
ecifi
c
auto
flu
ore
scen
cep
ho
tob
leac
hin
gch
arac
teri
stic
;au
tofl
uo
resc
ence
inte
nsi
tyo
fh
ealt
hy
skin
dec
reas
eex
po
nen
tial
ly;
auto
flu
ore
scen
ce
inte
nsi
tyo
fp
igm
ente
dn
evi
var
ies
aro
un
dth
ein
itia
lv
alu
e;p
igm
ente
d
cell
ula
rn
evu
san
dch
erry
ang
iom
ah
ave
dif
fere
nt
dy
nam
icfe
atu
res.
Res
ult
so
fth
ep
rese
nt
stu
dy
sho
wco
nsi
der
able
sen
siti
vit
yo
fsk
in
pat
ho
log
ies
of
the
auto
flu
ore
scen
cep
ho
tob
leac
hin
gan
aly
sis
met
ho
d
Lih
ach
evet
al.
(20
11
)
Vit
ilig
o
com
po
un
dn
evu
s
nev
us
of
Ota
mel
ano
ma
po
st-i
nfl
amm
ato
ry
hy
per
pig
men
tati
on
AF
TF
iber
-op
tic
NIR
spec
tro
met
er
k excit
ati
on
=7
85
nm
Tw
elv
ep
atie
nts
wit
h:
vit
ilig
o,
com
po
un
dn
evu
s,n
evu
so
fO
ta,
sup
erfi
cial
spre
adin
gm
elan
om
a,an
dp
ost
-in
flam
mat
ory
hy
per
pig
men
tati
on
wer
e
eval
uat
edb
yN
IR-A
FT
.T
he
resu
lts
of
this
stu
dy
sho
wed
that
all
thes
e
con
dit
ion
sex
hib
ited
sig
nifi
can
tly
gre
ater
NIR
flu
ore
scen
ceth
anth
e
surr
ou
nd
ing
no
rmal
skin
,ex
cep
tv
itil
igo
wh
ich
pre
sen
ted
alo
wer
auto
flu
ore
scen
ce.
Bas
edo
nth
ese
resu
lts,
the
auth
ors
con
clu
ded
that
NIR
flu
ore
scen
cete
chn
iqu
esco
uld
be
use
dto
eval
uat
eth
esk
ind
iso
rder
s
inv
olv
ing
mel
anin
Hu
ang
etal
.(2
00
6)
1088 J Cancer Res Clin Oncol (2013) 139:1083–1104
123
Ta
ble
2co
nti
nu
ed
Dis
ease
Flu
ore
scen
ce
tech
niq
ue
Dev
ice/
par
amet
ers
Res
ult
sR
efer
ence
Bas
alce
llca
rcin
om
aD
FT
Ph
oto
sen
siti
zer:
Met
hy
l
amin
ole
vu
lin
ate
(c=
16
0m
g/g
)
Dig
ital
flu
ore
scen
ceim
agin
gsy
stem
(Dy
aDer
m;
Bio
cam
Gm
bH
,
Reg
ensb
urg
,G
erm
any
)
k excit
ati
on
=4
05
nm
DP
=0
.8m
W/c
m2
k em
issi
on
-re
d(P
pIX
flu
ore
scen
ce)
k em
issi
on
-g
reen
(au
tofl
uo
resc
ence
)
Inth
isst
ud
y,
the
auth
ors
eval
uat
edth
ecl
inic
alp
erfo
rman
ceo
fa
pre
op
erat
ive
defi
nit
ion
of
the
late
ral
bo
rder
so
fB
CC
by
flu
ore
scen
ce
det
ecti
on
inco
mp
aris
on
wit
hit
sd
efin
itio
nb
ycl
inic
ald
iag
no
sis.
Th
e
mai
nre
sult
so
bta
ined
inth
isst
ud
yar
eas
foll
ow
s:th
em
ean
tum
or
area
as
det
erm
ined
by
flu
ore
scen
ced
etec
tio
nw
assi
gn
ifica
ntl
ysm
alle
rth
anth
e
tum
or
area
asd
eter
min
edb
ycl
inic
ald
iag
no
sis;
the
sen
siti
vit
yo
f
flu
ore
scen
ced
etec
tio
nw
as3
8.5
%;
the
spec
ifici
tyo
ffl
uo
resc
ence
det
ecti
on
was
calc
ula
ted
as8
8.4
%.
Bas
edo
nth
ese
resu
lts,
the
auth
ors
con
clu
de
that
pre
op
erat
ive
flu
ore
scen
ced
etec
tio
nco
mb
ined
wit
hcl
inic
al
dia
gn
osi
so
fn
od
ula
rB
CC
loca
lize
din
the
hig
h-r
isk
H-z
on
eh
asn
o
add
itio
nal
clin
ical
ben
efit
com
par
edw
ith
sim
ple
clin
ical
dia
gn
osi
sal
on
e
Wet
zig
etal
.(2
01
0)
Bas
alce
llca
rcin
om
aD
FT
Ph
oto
sen
siti
zer:
5-a
min
ole
vu
lin
ic
acid
(c=
20
%)
t incubati
on
=3
h
Dig
ital
flu
ore
scen
ceim
agin
gsy
stem
(Dy
aDer
m,
Bio
cam
Gm
bH
,
Reg
ensb
urg
,G
erm
any
)
Flu
ore
scen
ced
iag
no
sis
and
clin
ical
dia
gn
osi
sw
ere
use
din
this
stu
dy
as
met
ho
ds
for
the
no
nin
vas
ive
det
ecti
on
of
tum
or
bo
un
dar
ies.
Th
ere
sult
s
of
this
stu
dy
sho
wed
that
the
mea
ntu
mo
rar
eath
atw
asv
isu
aliz
edb
y
flu
ore
scen
ced
iag
no
sis
was
sig
nifi
can
tly
smal
ler
than
the
tum
or
area
det
erm
ined
by
clin
ical
dia
gn
osi
s(9
7.9
±3
4.7
mm
2v
s.
12
4.5
±3
7.6
mm
2).
Th
ese
resu
lts
lead
toth
eco
ncl
usi
on
that
flu
ore
scen
cete
chn
iqu
eis
less
sen
siti
ve
than
clin
ical
dia
gn
osi
so
fth
e
tum
or
bo
un
dar
ies
Gam
bic
hle
ret
al.
(20
08
)
Bas
alce
llca
rcin
om
aD
FT
Ph
oto
sen
siti
zer:
Met
hy
l
amin
ole
vu
lin
ate
–In
this
stu
dy
,th
ecl
inic
alef
fica
cyo
fP
pIX
flu
ore
scen
ceim
ages
usi
ng
flu
ore
scen
ceim
age
anal
ysi
sto
defi
ne
the
late
ral
bo
rder
bet
wee
nth
e
tum
or
and
tum
or-
free
area
so
ffa
cial
BC
Cw
asev
alu
ated
.T
he
rate
of
tum
or
det
ecti
on
fro
mB
CC
lesi
on
su
sin
gP
pIX
flu
ore
scen
cew
ith
the
flu
ore
scen
ceim
age
anal
ysi
sto
ol
sho
wed
ase
nsi
tiv
ity
of
94
.1%
and
spec
ifici
tyo
f8
2.6
%.
Th
ese
resu
lts
sug
ges
tth
atM
AL
-in
du
ced
Pp
IX
flu
ore
scen
ceim
agin
gu
sin
gfl
uo
resc
ence
imag
ean
aly
sis
isq
uit
ese
nsi
tiv
e
and
spec
ific
for
det
ecti
ng
tum
or
and
occ
ult
tum
or
infa
cial
BC
Cle
sio
ns
Wo
net
al.
(20
07
)
Bas
alce
llca
rcin
om
aD
FT
Ph
oto
sen
siti
zer:
5-a
min
ole
vu
lin
ic
acid
(c=
20
%)
t incubati
on
=3
.5h
Wo
od
lam
pT
he
flu
ore
scen
ced
iag
no
sis
and
his
top
ath
olo
gic
alex
amin
atio
nw
ere
use
d
inth
isst
ud
yas
met
ho
ds
for
the
det
ecti
on
of
tum
or
mar
gin
s.T
he
stu
dy
fin
din
gs
sho
wed
that
:
Insi
xB
CC
s,th
eD
FT
-defi
ned
BC
Cm
arg
ind
idn
ot
corr
elat
ew
ith
the
his
top
ath
olo
gic
ally
asse
ssed
tum
or
bo
rder
s;
Th
ese
nsi
tiv
ity
and
spec
ifici
tyra
tes
of
DF
Tw
ere
79
%an
d1
00
%,
resp
ecti
vel
y;
DF
Tis
fair
lyse
nsi
tiv
ean
dh
igh
lysp
ecifi
cm
eth
od
for
the
dem
arca
tio
no
f
BC
Cm
arg
ins;
DF
Td
oes
no
tse
emto
be
sub
stan
tial
lysu
per
ior
tosi
mp
lecl
inic
al
eval
uat
ion
of
tum
or
mar
gin
s
Neu
set
al.
(20
08
)
J Cancer Res Clin Oncol (2013) 139:1083–1104 1089
123
Ta
ble
2co
nti
nu
ed
Dis
ease
Flu
ore
scen
ce
tech
niq
ue
Dev
ice/
par
amet
ers
Res
ult
sR
efer
ence
Sq
uam
ou
sce
ll
carc
ino
ma
DF
T
Ph
oto
sen
siti
zer:
Rad
ach
lori
n
c=
1.0
mg
/kg
t incubati
on
=4
–5
h
Flu
ore
scen
ceim
agin
gsy
stem
(Flu
ote
st)
k excit
ati
on
=6
33
nm
P=
10
0m
W
Th
eac
cura
cyo
fD
FT
for
the
dia
gn
osi
so
fS
CC
was
inv
esti
gat
edin
40
pat
ien
ts.
Th
ere
sult
so
ffl
uo
resc
ence
and
his
top
ath
olo
gic
alst
ud
ies
sim
ilar
lysh
ow
ed:
mal
ign
ant
lesi
on
(in
27
case
s)an
dn
on
-mal
ign
ant
lesi
on
(in
8ca
ses)
.In
5ca
ses,
the
resu
lts
of
thes
etw
om
eth
od
sw
ere
dif
fere
nt.
Th
ep
erfo
rman
ces
of
DF
Tin
dia
gn
osi
ng
SC
Cw
ere
also
eval
uat
ed:
90
%—
sen
siti
vit
y;
80
%—
spec
ifici
ty;
87
.5%
—ac
cura
cy;
93
%—
po
siti
ve
pre
dic
tiv
ev
alu
e(P
PV
);7
2%
—n
egat
ive
pre
dic
tiv
e
val
ue
(NP
V);
4.5
—p
osi
tiv
eli
kel
iho
od
rati
o(P
LR
)an
d0
.12
5—
neg
ativ
e
lik
elih
oo
dra
tio
(NL
R).
Th
ese
resu
lts
pro
ve
bo
thac
cura
cyan
dre
liab
ilit
y
of
DF
Tm
eth
od
for
det
ecti
ng
SC
Cle
sio
ns
Kam
rav
aet
al.
(20
12
)
Sq
uam
ou
sce
ll
carc
ino
ma
Bas
alce
llca
rcin
om
a
Ad
eno
carc
ino
ma
Ch
on
dro
sarc
om
a
DF
T
Ph
oto
sen
siti
zers
:
Hem
ato
po
rph
yri
n
der
ivat
ive
(c=
2.5
–5
mg
/kg
)
t incubati
on
=1
2–
24
h
5-a
min
ole
vu
lin
ic
acid
(c=
20
%)
t incubati
on
=2
–8
h
Sy
stem
bas
edo
nb
lue
lig
ht–
emit
tin
g
dio
des
k excit
ati
on
=(3
78
–4
26
)n
m
P=
1–
10
mW
Nin
ety
-eig
ht
pat
ien
tsw
ith
mal
ign
ant,
pre
mal
ign
ant,
and
ben
ign
skin
wer
e
inv
esti
gat
edb
yD
FT
.T
he
stu
dy
hig
hli
gh
ted
the
foll
ow
ing
:
Th
etu
mo
rsm
arg
ins
can
be
clea
rly
and
pre
cise
lyo
utl
ined
un
der
flu
ore
scen
tv
isio
n;
Th
em
ost
app
rop
riat
ew
avel
eng
thfo
rD
FT
is4
01
nm
ino
rder
toac
hie
ve
com
ple
tev
isu
aliz
atio
no
fm
alig
nan
tle
sio
ns
afte
rth
eap
pli
cati
on
of
a
tum
or
sele
ctiv
ep
ho
tose
nsi
tize
r;
Inth
eb
lue
lig
ht
mo
de,
ther
eis
bac
kg
rou
nd
blu
efl
uo
resc
ence
inn
orm
al
tiss
ue
and
red
flu
ore
scen
cein
mal
ign
ant
area
s.T
he
auth
ors
con
clu
ded
that
DF
Tis
app
lica
ble
for
det
ecti
ng
earl
ysu
per
fici
altu
mo
rs
Liu
tkev
iciu
te-
Nav
ick
ien
eet
al.
(20
08)
Seb
aceo
us
Gla
nd
hy
per
pla
sia
Act
inic
ker
ato
sis
Bas
alce
llca
rcin
om
a
Sq
uam
ou
sce
ll
carc
ino
ma
DF
T
Ph
oto
sen
siti
zers
:
5-a
min
ole
vu
lin
ic
acid
(c=
0.5
%)
Met
hy
l
amin
ole
vu
lin
ate
(c=
16
%)
t incubati
on
=3
h
Dy
aDer
mfl
uo
resc
ence
det
ecti
on
syst
em
Lig
ht
sou
rce:
LE
D
k=
40
5n
m
s=
5m
s
m=
1H
z
P=
1.0
W
Six
ty-o
ne
pat
ien
tsw
ith
28
7le
sio
ns
(21
2b
enig
nle
sio
ns,
71
pre
mal
ign
ant
lesi
on
s,3
BC
C,
and
1S
CC
)w
ere
inv
esti
gat
edin
this
stu
dy
by
DF
T,
usi
ng
AL
Ao
rM
AL
asp
ho
tose
nsi
tize
r.T
he
flu
ore
scen
cein
ten
siti
eso
f
lesi
on
sw
ere
eval
uat
edin
com
par
iso
nw
ith
the
his
top
ath
olo
gic
al
exam
inat
ion
.T
he
flu
ore
scen
ceim
age
of
MA
L-t
reat
edsk
inar
eas
sho
wed
ver
yh
igh
and
ho
mo
gen
eou
sfl
uo
resc
ence
inte
nsi
tyw
ith
low
dis
crim
inat
ion
bet
wee
nn
orm
alan
dd
isea
sed
skin
.T
he
AL
A-t
reat
ed
area
ssh
ow
edlo
wau
tofl
uo
resc
ence
of
the
no
rmal
skin
and
mo
der
ate,
bu
t
dis
tin
ctfl
uo
resc
ence
of
acti
nic
ker
ato
ses,
resu
ltin
gin
ah
igh
dis
crim
inat
ion
bet
wee
nth
en
orm
alan
dth
ed
isea
sed
skin
.T
he
resu
lts
of
this
stu
dy
rev
eale
dth
atth
esp
ecifi
city
of
com
bin
edm
eth
od
(DF
T,
clin
ical
inv
esti
gat
ion
,an
dd
erm
ato
sco
py
)is
abo
ut
92
%
Lee
uw
etal
.(2
00
9)
Act
inic
ker
ato
sis,
mo
rbu
sB
ow
en,
bas
alce
ll
carc
ino
ma
Ben
ign
lesi
on
s
DF
T
Ph
oto
sen
siti
zer:
5-a
min
ole
vu
lin
ic
acid
(c=
0.5
%)
t incubati
on
=2
.5h
Dy
ader
mfl
uo
resc
ence
det
ecti
on
syst
em
(fo
rmer
lyB
ioca
mG
mb
H,
Reg
ensb
urg
,
FR
G).
k=
40
7n
m
Th
est
ud
yw
asco
nd
uct
edo
n3
0p
atie
nts
susp
ecte
do
fh
avin
go
ne
or
mo
re
no
n-m
elan
om
ask
inca
nce
rs(N
MS
C).
Aco
mp
aris
on
bet
wee
nth
e
accu
racy
of
no
n-n
orm
aliz
edan
dn
orm
aliz
edfl
uo
resc
ence
met
ho
ds
was
do
ne.
Th
ere
sult
so
fth
isst
ud
yre
vea
led
that
the
spec
ifici
tyan
dse
nsi
tiv
ity
of
no
n-n
orm
aliz
edfl
uo
resc
ence
met
ho
dar
esu
bst
anti
ally
low
erth
an
tho
seo
fn
orm
aliz
edfl
uo
resc
ence
det
ecti
on
met
ho
d(2
7an
d3
9%
vs.
10
0
and
97
%)
van
der
Bee
ket
al.
(20
12)
1090 J Cancer Res Clin Oncol (2013) 139:1083–1104
123
2012), while other researchers concluded that DFT had no
value in early skin cancer detection in premalignant lesions
(Kleinpenning et al. 2010; van der Beek et al. 2012). Some
studies insisted only in comparison between photosensi-
tizers (Liutkeviciute-Navickiene et al. 2008) or the best
way to analyze data [normalized fluorescence detection
showing better results (Leeuw et al. 2009)].
Nevertheless, the fluorescence spectroscopy offers the
advantage that it can directly probe the biochemical
composition of tissues by means of detecting specific
biomolecules which emits characteristic fluorescence sig-
nals. Furthermore, fluorescence is very sensitive to factors
such as pH and temperature in addition to other uncon-
trollable physiological factors that induce wide variability
to the data. Having the huge advantage of being a nonin-
vasive method, fluorescence, both natural and drug-
induced, has not shown constant results in determining
early malignancy, nor in tumor margin assessment, which
calls for further studies concerning its clinical value.
Diffuse reflectance spectroscopy (DRS)
Diffuse reflectance spectroscopy is a potentially affordable
technique that can be used for fast, noninvasive and accu-
rate diagnosis of skin disease. This technique is sensitive to
both scattering and absorption properties of the tissue, over
a wide range of wavelengths, and consequently, it can
provide spectra that contain valuable information about the
morphology of the normal or abnormal tissue as well as the
chromophore content (e.g., hemoglobin, melanin, bilirubin,
and water). The use of DRS for tissue diagnosis is based on
the fact that many tissue pathologies exhibit significant
architectural changes at the cellular and subcellular levels
which can be evidenced by spectral measurements of the
diffuse reflected light.
The principle of diffuse reflectance spectrometry con-
sists in sending a light beam toward a sample and detecting
light reflected from its surface in many directions in the
hemisphere surrounding the surface. The general mecha-
nism by which the skin reflects light diffusely does not
involve only the skin surface, but also the presence of
scattering centers located below the skin surface. By
measuring the changes in the diffuse reflectance spectrum,
information about changes of scattering centers (and thus
of the specific structures of the skin) can be obtained.
Unlike the fluorescence techniques, DRS does not provide
information about the chemical composition of tissue in a
direct way, but using some analytical models of light
transport in biological tissues (Zonios et al. 2001) or
numerical methods (Wang and Jacques 1995; Yudovsky
and Laurent Pilon 2010), some skin constituents can be
determined (e.g., hemoglobin and melanin). Hence, DRS
which provides important data about both structure andTa
ble
2co
nti
nu
ed
Dis
ease
Flu
ore
scen
ce
tech
niq
ue
Dev
ice/
par
amet
ers
Res
ult
sR
efer
ence
Act
inic
ker
ato
sis
squ
amo
us
cell
carc
ino
ma
(SC
C)
DF
T
Ph
oto
sen
siti
zer:
Met
hy
l
amin
ole
vu
lin
ate
t incubati
on
=3
h
Dig
ital
flu
ore
scen
ce
imag
ing
syst
em(D
yad
erm
,B
ioca
m
Gm
bH
,R
egen
sbu
rg,
Ger
man
y)
Inth
ep
rese
nt
stu
dy
,th
ep
ote
nti
alap
pli
cab
ilit
yo
fD
FT
ind
iscr
imin
atin
g
Ak
sfr
om
SC
Cw
asin
ves
tig
ated
.
Th
ele
sio
nal
/no
n-l
esio
nal
flu
ore
scen
cera
tio
of
Ak
sw
asco
mp
ared
wit
hth
e
rati
oo
fS
CC
.1
3p
atie
nts
wit
h3
6le
sio
ns
susp
ecte
dfo
rA
Ko
rS
CC
wer
e
incl
ud
edin
this
stu
dy
.A
llle
sio
ns
wer
ed
iag
no
sed
by
DF
Tan
d
his
top
ath
olo
gic
alex
amin
atio
n.
No
sig
nifi
can
td
iffe
ren
ces
wer
efo
un
din
the
flu
ore
scen
cera
tio
(les
ion
al/n
on
-les
ion
alsk
in)
bet
wee
nA
ks
and
SC
Cs,
alth
ou
gh
mac
rosc
op
icfl
uo
resc
ence
was
sig
nifi
can
tly
hig
her
in
Bo
wen
’sd
isea
sean
dm
icro
inv
asiv
eS
CC
s
Kle
inp
enn
ing
etal
.
(20
10)
J Cancer Res Clin Oncol (2013) 139:1083–1104 1091
123
chemical composition of tissue is now considered to be an
appropriate technique for early diagnosis of some disease,
especially premalignant or malignant skin lesions
(Table 3), having the advantage of avoiding tissue biopsy
and providing diagnostic signatures, noninvasively and in
real time.
According to Table 3, the values of sensitivity and
specificity of the DRS technique strongly depend on the
specific features of the spectra obtained from normal,
precancerous, and cancerous skin lesions taken into
account in discriminant analysis used by different
researchers. Such specific features of the skin spectra
reported to date in differentiating between normal and
pathological tissue are mean or integral value of absorption
coefficient and reduced scattering coefficient (Zonios et al.
2008; Garcia-Uribe et al. 2011), spectral slope (Canpolat
et al. 2012), and integral value of the diffuse reflectance for
specific wavelength region (Canpolat et al. 2007; Jiao et al.
2009; Upile et al. 2012). Based on the comparison between
these specific spectral features, the values of the specificity
and sensitivity of DRS were established to be between
72–92 % and 64–92 %, respectively.
Recently, several research groups have demonstrated
that these performances of DRS in differentiating disease
from normal surrounding tissue, mainly in detection of
cancerous and precancerous changes in human skin can be
improved by combining this noninvasive diagnostic tech-
nique with AFT. Using this combined technique (DRS/
AFT), some parameters related to the biochemical, archi-
tectural, and morphologic state of tissue can be simulta-
neously measured and used to diagnose various skin
conditions. Thus, Thompson et al. (2012) using a compact
steady-state diffuse reflectance/fluorescence spectrometer
and a fiber-optic-coupled multispectral time-resolved
spectrofluorometer have correctly diagnosed 87 % of the
BCCs in 25 patients. Very good results were also reported
by Rajaram et al. (2010) in a pilot clinical study performed
on 40 patients with 48 lesions. They have demonstrated
that, using a combined method (DRS/AFT), BCCs can be
classified with a sensitivity and specificity of 94 and 89 %,
respectively, while actinic keratoses and squamous cell
carcinomas with a sensitivity of 100 % and specificity of
50 %. Troyanova et al. (2007) reported that the differen-
tiation between normal skin and different cutaneous lesion
types (hemangioma, angiokeratoma, and fibroma) and
among lesion types themselves can be done with the sen-
sitivities and specificities higher than 90 % by common use
of laser-induced autofluorescence (LIAF) and reflectance
spectroscopy (DRS). The same technique (LIAF/DRS) was
also used by Borisova et al. (2012) for skin cancer diag-
nostic. They reported a sensitivity of 92 % and specificity
of 78 % of combined LIAF/DRS technique in discrimina-
tion between malignant melanoma from dysplastic nevi.
All these results demonstrate that the diagnostic accuracy
can be improved by the use of combined technique (AFT/
DRS) together the specific discriminant analysis.
Besides these spectroscopic techniques (DRS and fluo-
rescence spectroscopy), Raman spectroscopy has also been
used for the same medical applications.
Raman spectroscopy
Raman spectroscopy is an optical technique which uses the
inelastic scattering of monochromatic light (usually with
wavelength in the visible, near-infrared, or near-ultraviolet
range) to analyze vibrational modes of molecules. The
inelastic light scattering process occurs when the photons
interact with the vibrating molecules or the excited elec-
trons in the sample in such a way that molecules take up
energy from or give up energy to the photons, so that the
scattered photons are shifted in frequency up or down in
comparison with the incident photons. The shift in the
photon frequency is correlated with the difference between
initial and final vibrational energy levels of the scattering
molecule. The change in energy or shift in photon fre-
quency indicates molecular information and its photon
mode in the sample. These changes in energy and fre-
quency are molecular-specific and they appear as a series
of peaks in a Raman spectrum. The positions and relative
magnitudes of these peaks correspond to the vibrational
energies associated with specific chemical bonds in specific
molecules. Many molecules have distinguishable spectra,
so that one can determine the molecular composition of a
sample from its Raman spectrum.
Raman spectroscopy has a wide range of uses in various
biomedical issues such as early detection of neoplastic
lesions (Qiang and Chang 2012), intraoperative tumor
border determination (Haka et al. 2006; Keller et al. 2011),
determination of atherosclerotic plaque composition (Motz
et al. 2006; Rocha et al. 2007), assessment of the chemical
substance toxicity (Pyrgiotakis et al. 2009), and identifi-
cation of pathogenic microorganisms (Kalasinsky et al.
2007; de Siqueira et al. 2012). since it can provide details
of the molecular and/or biochemical changes associated
with the morphological changes that occur in tissue as a
result of disease.
In dermatology, RS has beginning to be recognized as a
potential technique for the diagnosis of skin cancer and
characterization of neoplastic progression of tissues with a
high degree of specificity at the molecular level (Table 4).
As we can see in Table 4, RS can be applied both in vivo
and on tissue samples (Zhao et al. 2008; Zeng et al. 2011; Lui
et al. 2012). Both skin carcinomas and melanomas have been
taken into account during research, with fewer studies con-
centrating on only one type of skin malignancy (Nunes et al.
2003; Zhao et al. 2008; Larraona-Puy et al. 2009). The
1092 J Cancer Res Clin Oncol (2013) 139:1083–1104
123
Ta
ble
3C
lin
ical
app
lica
tio
ns
of
dif
fuse
refl
ecta
nce
spec
tro
met
ryfo
rth
ed
iag
no
sis
of
skin
dis
ease
s
Dis
ease
Dev
ice/
par
amet
ers
Res
ult
sR
efer
ence
Mel
ano
ma
Dy
spla
stic
nev
i
Co
mm
on
nev
i
Ob
liq
ue
inci
den
ced
iffu
sere
flec
tan
cesp
ectr
osc
op
ic(O
IDR
S)
syst
em:
Lig
ht
sou
rce:
hal
og
enla
mp
(45
5–
76
5)
nm
;
Fib
er-o
pti
cse
nso
rp
rob
e:o
ne
or
mo
reso
urc
efi
ber
for
a4
5�
ob
liq
ue
inci
den
cean
d2
lin
ear
arra
yo
f1
0co
llec
tio
nfi
ber
s
Inth
isst
ud
y,
DR
Sw
asap
pli
edo
n1
44
pig
men
ted
skin
lesi
on
s(1
6
mel
ano
mas
,9
8d
ysp
last
icn
evi,
and
30
com
mo
nn
evi)
.T
he
op
tica
l
abso
rpti
on
and
scat
teri
ng
spec
tra
of
thes
esk
inle
sio
ns
wer
e
esti
mat
edb
yth
eau
tho
rso
fth
isst
ud
yfr
om
the
mea
sure
dd
iffu
se
refl
ecta
nce
dat
a.T
he
resu
lts
rev
eale
dth
atth
eab
sorp
tio
nsp
ectr
a
for
the
mel
ano
ma
and
dy
spla
stic
case
sar
esi
mil
aran
dg
ener
ally
are
hig
her
than
tho
sefo
rth
eb
enig
no
nes
.A
lso
,it
was
fou
nd
that
the
red
uce
dsc
atte
rin
gco
effi
cien
tg
ener
ally
incr
ease
sw
ith
the
deg
ree
of
dy
spla
sia
or
mal
ign
ancy
of
the
skin
lesi
on
sw
ith
inth
e
enti
rev
isib
lesp
ectr
um
Gar
cia-
Uri
be
etal
.(2
01
1)
Mel
ano
mas
dy
spla
stic
nev
ico
mm
on
nev
i
Bas
alce
llca
rcin
om
a
squ
amo
us
cell
carc
ino
ma
Ob
liq
ue
inci
den
ced
iffu
sere
flec
tan
cesp
ectr
osc
op
ic(O
IDR
S)
syst
em
Inth
isst
ud
y,
the
OID
RS
syst
emw
asu
sed
for
the
dia
gn
osi
so
f
mel
ano
ma
and
no
n-m
elan
om
ask
inca
nce
r.T
he
resu
lts
sho
wed
that
pig
men
ted
mel
ano
ma
was
dia
gn
ose
dw
ith
sen
siti
vit
yan
d
spec
ifici
tyo
f9
0%
for
ab
lin
ded
test
set.
Th
ese
nsi
tiv
ity
and
spec
ifici
tyo
fD
RS
met
ho
dh
ave
incr
ease
dto
92
%in
the
case
of
dif
fere
nti
atio
no
fn
on
-pig
men
ted
bas
alce
llo
rsq
uam
ou
sce
ll
carc
ino
mas
fro
mn
on
-can
cero
us
skin
abn
orm
alit
ies
(act
inic
ker
ato
ses
and
seb
orr
hei
ck
erat
ose
s)
Gar
cia-
Uri
be
etal
.(2
01
2)
Mel
ano
ma
Co
mm
on
nev
us
Dy
spla
stic
nev
us
Fib
er-o
pti
cp
rob
e:4
00
lm
core
mu
ltim
od
efi
ber
sar
ran
ged
ina
6il
lum
inat
ion
aro
un
d1
coll
ecti
on
geo
met
ryw
ith
asi
ng
le
fib
er–
fib
ersp
acin
go
f4
70
lm
k[
(55
0–
1,0
00
)n
m
Th
est
ud
yas
sess
edd
iscr
imin
atio
no
fea
rly
mel
ano
ma
fro
mco
mm
on
and
dy
spla
stic
nev
us,
usi
ng
DR
S.
Th
est
ud
yw
asco
nd
uct
edo
n
12
0p
igm
ente
dle
sio
ns
of
wh
ich
64
wer
eco
nfi
rmed
his
top
ath
olo
gic
ally
,as
mel
ano
ma.
As
are
sult
,th
ev
aria
tio
ns
in
spec
tra
bet
wee
ng
rou
ps
of
lesi
on
sw
ith
dif
fere
nt
dia
gn
ose
sw
ere
exam
ined
and
red
uce
dto
feat
ure
ssu
itab
lefo
rd
iscr
imin
ant
anal
ysi
s.A
clas
sifi
erd
isti
ng
uis
hin
gb
etw
een
ben
ign
and
mal
ign
ant
lesi
on
sw
asp
erfo
rmed
wit
hse
nsi
tiv
ity
/sp
ecifi
city
of
bet
wee
n
64
–6
9%
and
72
–7
8%
.T
hes
ere
sult
sd
emo
nst
rate
dth
atcl
assi
fier
s
bet
wee
np
airs
of
the
gro
up
com
mo
nn
evu
s,d
ysp
last
icn
evu
s,
insi
tum
elan
om
a,an
din
vas
ive
mel
ano
ma
sho
wb
ette
ro
rsi
mil
ar
per
form
ance
than
the
ben
ign
/mal
ign
ant
clas
sifi
er
Mu
rph
yet
al.
(20
05
)
Mel
ano
ma
Dy
spla
stic
nev
i
Sp
ectr
op
ho
tom
eter
(Oce
anO
pti
cs,
SB
20
00
)
Lig
ht
sou
rce:
tun
gst
en–
hal
og
enli
gh
tso
urc
e(O
cean
Op
tics
,
HL
-20
00
);
Fib
er-o
pti
cp
rob
e:6
con
cen
tric
ally
arra
ng
ed2
00
lm
core
op
tica
lfi
ber
s,an
dfo
rd
iffu
sere
flec
tan
ceco
llec
tio
n,
asi
ng
le
20
0l
mco
rece
ntr
alo
pti
cal
fib
er(O
cean
Op
tics
,R
20
0-7
)
k[
(46
0–
1,0
00
)n
m
Th
est
ud
yw
asco
nd
uct
edo
n1
0h
um
ansu
bje
cts
incl
ud
ing
6w
ith
dy
spla
stic
nev
i,1
wit
hm
elan
om
ain
situ
,an
d3
wit
hm
alig
nan
t
mel
ano
ma.
Th
ere
sult
so
fth
isst
ud
yd
emo
nst
rate
dth
atth
eo
pti
cal
abso
rpti
on
spec
tru
mo
fin
viv
om
elan
inp
rese
nts
anex
po
nen
tial
dep
end
ence
on
wav
elen
gth
.T
he
auth
ors
just
ify
theo
reti
call
yth
is
exp
on
enti
ald
epen
den
ceo
nth
eb
asis
of
are
cen
tly
pro
po
sed
mo
del
for
the
stru
ctu
reo
feu
mel
anin
pro
tom
ole
cule
s.A
lso
,a
new
met
ho
dfo
ran
aly
zin
gd
iffu
sere
flec
tan
cesp
ectr
a,w
hic
hid
enti
fies
intr
insi
cd
iffe
ren
ces
inab
sorp
tio
nsp
ectr
ab
etw
een
mal
ign
ant
mel
ano
ma
and
dy
spla
stic
nev
iin
viv
o,
was
rep
ort
ed.
Th
eau
tho
rs
hav
eal
sofo
un
dev
iden
ceth
atth
eh
isto
log
ictr
ansi
tio
nfr
om
dy
spla
stic
nev
ito
mel
ano
ma
insi
tuan
dth
ento
mal
ign
ant
mel
ano
ma
isre
flec
ted
inth
em
elan
inab
sorp
tio
nsp
ectr
a
Zo
nio
set
al.
(20
08
)
J Cancer Res Clin Oncol (2013) 139:1083–1104 1093
123
Ta
ble
3co
nti
nu
ed
Dis
ease
Dev
ice/
par
amet
ers
Res
ult
sR
efer
ence
Mel
ano
ma
Bas
alce
llca
rcin
om
a
squ
amo
us
cell
carc
ino
ma
Ben
ign
lesi
on
s
–T
wen
ty-t
hre
ep
atie
nts
wit
htw
enty
-eig
ht
lesi
on
s(9
BC
C,
4
mel
ano
ma,
2S
CC
,an
d1
3b
enig
nle
sio
ns)
wer
ein
ves
tig
ated
in
this
stu
dy
by
DR
S.
Als
o,
intr
aop
erat
ive
mar
gin
asse
ssm
ents
wer
e
also
per
form
edo
nth
e2
8b
iop
sysa
mp
les.
Th
ere
sult
so
fth
est
ud
y
sho
wed
that
the
sig
no
fth
esp
ectr
alsl
op
ew
asp
osi
tiv
efo
rb
enig
n
and
neg
ativ
efo
rm
alig
nan
tti
ssu
es.
Th
isp
aram
eter
was
use
dto
dis
crim
inat
eb
etw
een
mal
ign
ant
and
ben
ign
lesi
on
sw
ith
a
sen
siti
vit
yan
dsp
ecifi
city
of
87
and
85
%,
resp
ecti
vel
y.
Sen
siti
vit
yan
dsp
ecifi
city
of
the
syst
emin
det
ecti
ng
po
siti
ve
surg
ical
mar
gin
so
n1
4ex
cise
db
iop
sysa
mp
les
wer
e8
0an
d9
0%
,
resp
ecti
vel
y
Can
po
lat
etal
.(2
01
2)
Bas
alce
llca
rcin
om
a
seb
orr
hei
ck
erat
osi
s
fib
roep
ith
elia
lp
oly
p
Intr
ader
mal
nev
i
–T
he
stu
dy
was
con
du
cted
on
73
pat
ien
tsw
ith
faci
alsk
inle
sio
ns
(bas
alce
llca
rcin
om
a,se
bo
rrh
eic
ker
ato
sis,
fib
roep
ith
elia
lp
oly
p,
and
intr
ader
mal
nev
i).
Th
ere
sult
so
fth
est
ud
yre
vea
led
that
DR
S
can
dif
fere
nti
ate
bet
wee
nn
orm
alan
dp
ath
olo
gic
alsk
inco
nd
itio
ns
asw
ell
asb
enig
nan
dm
alig
nan
tsk
inco
nd
itio
ns
Jiao
etal
.(2
00
9)
Bas
alce
llca
rcin
om
aD
iffu
sere
flec
tan
cesp
ectr
osc
op
icsy
stem
Lig
ht
sou
rce:
pu
lsed
xen
on
-arc
lam
p
Op
tica
lfi
ber
pro
be:
on
eil
lum
inat
ion
fib
er(4
00
lm
)an
do
ne
fib
erw
hic
hco
llec
tth
eli
gh
tre
flec
ted
(20
0lm
)
k[
(33
0–
75
0)
nm
Sev
enty
-th
ree
pat
ien
tsw
ere
inv
esti
gat
edin
this
stu
dy
by
DR
S.
Co
mp
aris
on
of
the
his
tolo
gic
ald
iag
no
sis
and
DR
Sin
the
dia
gn
osi
so
fB
CC
resu
lted
ina
sen
siti
vit
y7
7.8
%an
dsp
ecifi
city
80
.3%
Up
ile
etal
.(2
01
2)
Mal
ign
skin
lesi
on
sD
iffu
sere
flec
tan
cesp
ectr
osc
op
icsy
stem
:
UV
spec
tro
met
er
Sin
gle
op
tica
lfi
ber
pro
be
Eig
hte
enp
atie
nts
wit
htw
enty
lesi
on
sw
ere
inv
esti
gat
edin
this
stu
dy
by
DR
San
dsk
inb
iop
sy
Th
ere
sult
sh
ave
dem
on
stra
ted
ag
oo
dco
rrel
atio
nb
etw
een
dif
fuse
refl
ecta
nce
spec
tra
and
the
pat
ho
log
yre
sult
sw
ith
sen
siti
vit
yan
d
spec
ifici
tyo
f8
2an
d8
9%
,re
spec
tiv
ely
Can
po
lat
etal
.(2
00
7)
1094 J Cancer Res Clin Oncol (2013) 139:1083–1104
123
Ta
ble
4A
pp
lica
tio
no
fR
aman
spec
tro
sco
py
for
the
dia
gn
osi
so
fsk
inca
nce
r
Dis
ease
Dev
ice/
par
amet
ers
Res
ult
sR
efer
ence
Mel
ano
ma
Ram
ansp
ectr
om
eter
syst
emR
aman
spec
tra
of
28
9sk
inca
nce
rsan
db
enig
nsk
inle
sio
ns
wer
ean
aly
zed
usi
ng
par
tial
leas
t-
squ
ares
reg
ress
ion
and
lin
ear
dis
crim
inan
tan
aly
sis.
Th
ere
sult
sh
ave
rev
eale
dth
atsk
in
can
cers
cou
ldb
ew
ell
dif
fere
nti
ated
fro
mb
enig
nsk
inle
sio
ns
(sen
siti
vit
y9
1%
and
spec
ifici
ty
75
%)
and
mal
ign
ant
mel
ano
ma
fro
mb
enig
np
igm
ente
dle
sio
ns
(sen
siti
vit
y9
7%
,sp
ecifi
city
78
%)
Zh
aoet
al.
(20
08
)
Mel
ano
ma
Bas
alce
llca
rcin
om
a
Sq
uam
ou
sce
llca
rcin
om
a
Act
inic
ker
ato
ses
Aty
pic
aln
evi
Mel
ano
cyti
cn
evi
Blu
en
evi
Seb
orr
hei
ck
erat
ose
s
Inte
gra
ted
real
-tim
esy
stem
of
Ram
ansp
ectr
osc
op
y
Th
est
ud
yw
asp
erfo
rmed
on
45
3p
atie
nts
wit
h5
18
ben
ign
and
mal
ign
ant
skin
lesi
on
s.T
he
resu
lts
rev
eale
dth
atR
aman
spec
tro
sco
py
can
be
use
dto
dis
tin
gu
ish
mal
ign
ant
fro
mb
enig
n
skin
lesi
on
sw
ith
go
od
dia
gn
ost
icac
cura
cy.
Th
ese
nsi
tiv
ity
of
the
met
ho
dw
asre
po
rted
tob
e
bet
wee
n9
5an
d9
9%
,w
hil
esp
ecifi
city
ran
ges
fro
m1
5an
d5
4%
Lu
iet
al.
(20
12
)
Mel
ano
ma
Pig
men
ted
nev
i
Bas
alce
llca
rcin
om
a
Seb
orr
hei
ck
erat
ose
s
NIR
-FT
-Ram
ansp
ectr
om
eter
Inth
isp
aper
,th
eau
tho
rsin
ves
tig
ated
the
po
ssib
ilit
yo
fu
sin
gch
emic
alch
ang
esin
the
mel
ano
ma
tiss
ue
det
ecte
db
yR
aman
spec
tro
sco
py
and
neu
ral
net
wo
rks
for
dia
gn
ost
ic
pu
rpo
ses.
Inth
isre
spec
t,F
T-R
aman
spec
tra
fro
msa
mp
les
of
mel
ano
ma
(n=
22
)w
ere
com
par
edto
pig
men
ted
nev
i(n
=4
1),
BC
C(n
=4
8),
seb
orr
hei
ck
erat
ose
s(n
=2
3)
usi
ng
neu
ral
net
wo
rkan
aly
sis.
Th
ere
sult
sre
vea
led
that
the
ban
dal
tera
tio
ns
(am
ide
Ip
rote
inb
and
(1,6
60
cm-
1)
and
lip
id-s
pec
ific
ban
dp
eak
s(1
,31
0an
d1
,33
0cm
-1)
can
be
ind
epen
den
tly
iden
tifi
edb
yn
eura
ln
etw
ork
anal
ysi
so
fR
aman
spec
tra.
Th
eau
tho
rsre
po
rta
sen
siti
vit
yan
d
spec
ifici
tyo
fth
ism
eth
od
for
the
dia
gn
osi
so
fm
elan
om
ao
f8
5an
d9
9%
,re
spec
tiv
ely
Gn
iad
eck
aet
al.
(20
04
)
Mel
ano
ma
Bas
alce
llca
rcin
om
a
Sq
uam
ou
sce
llca
rcin
om
a
Act
inic
ker
ato
ses
Seb
orr
hei
ck
erat
ose
s
Var
iou
sn
evi
AR
aman
spec
tro
sco
py
stu
dy
was
carr
ied
ou
to
n2
74
skin
lesi
on
sin
clu
din
gca
nce
rs(3
1
mel
ano
mas
,1
8b
asal
cell
carc
ino
mas
,an
d3
9sq
uam
ou
sce
llca
rcin
om
as),
pre
can
cero
us
lesi
on
s(2
0ac
tin
ick
erat
ose
s),
and
ben
ign
lesi
on
s(4
8se
bo
rrh
eic
ker
ato
ses
and
11
8v
ario
us
nev
i).
Th
ere
sult
of
this
stu
dy
sho
wed
that
pre
can
cero
us
lesi
on
san
dca
nce
rou
sle
sio
ns
can
be
dif
fere
nti
ated
fro
mb
enig
nle
sio
ns
wit
ha
sen
siti
vit
yo
f9
0%
and
spec
ifici
tyo
f7
5%
.T
hes
e
resu
lts
can
pro
vid
eth
eb
asis
for
intr
od
uci
ng
Ram
ansp
ectr
osc
op
yto
clin
ical
use
Zen
get
al.
(20
11
)
Mel
ano
ma
Pig
men
ted
nev
i
FT
-Ram
ansp
ectr
om
eter
(Bru
ker
RF
S1
00
,K
arls
ruh
e,G
erm
any
)
Ex
cita
tio
nso
urc
e:N
d:Y
AG
lase
r
k=
1,0
64
nm
P=
30
0m
W
reso
luti
on
=4
cm-
1
Inth
isst
ud
y,
10
sam
ple
so
fcu
tan
eou
sm
elan
om
a,9
sam
ple
so
fp
igm
ente
dn
evi,
and
10
sam
ple
s
of
no
rmal
skin
wer
ein
ves
tig
ated
by
FT
-Ram
ansp
ectr
osc
op
yin
ord
erto
dif
fere
nti
ate
cuta
neo
us
mel
ano
ma
and
pig
men
ted
nev
us.
Th
ere
sult
so
fth
isst
ud
yh
ave
sho
wn
that
the
no
rmal
skin
did
no
tsh
ow
asi
gn
ifica
nt
var
iati
on
bet
wee
nth
esp
ectr
a;th
esp
ectr
alst
and
ard
of
the
pig
men
ted
nev
ig
rou
psh
ow
edsi
gn
ifica
nt
var
iati
on
,an
dth
ecu
tan
eou
sm
elan
om
ag
rou
p
also
sho
wed
var
iati
on
.T
he
dis
crim
inat
ory
anal
ysi
so
fth
eR
aman
spec
tra
sho
wed
a7
5.3
%
effi
cien
cyo
fth
ed
iffe
ren
tiat
ion
bet
wee
nth
eth
ree
gro
up
sst
ud
ied
Car
tax
oet
al.
(20
10
)
J Cancer Res Clin Oncol (2013) 139:1083–1104 1095
123
Ta
ble
4co
nti
nu
ed
Dis
ease
Dev
ice/
par
amet
ers
Res
ult
sR
efer
ence
Mel
ano
ma
Ly
mp
hn
od
em
etas
tasi
s
FT
-Ram
ansp
ectr
om
eter
(Bru
ker
RF
S1
00
/S,
Kar
lsru
he,
Ger
man
y)
Ex
cita
tio
nso
urc
e:N
d:Y
AG
lase
r
k=
1,0
64
nm
P=
30
0m
W
Res
olu
tio
n=
4cm
-1
Ato
tal
of
37
1R
aman
spec
tra
fro
m1
0n
orm
alh
um
ansk
insa
mp
les
(10
5sp
ectr
a),
10
cuta
neo
us
mel
ano
ma
frag
men
ts(1
40
spec
tra)
,an
d9
lym
ph
no
de
met
asta
sis
sam
ple
s(1
26
spec
tra)
wer
e
acq
uir
edan
dan
aly
zed
inth
isst
ud
yin
ord
erto
ob
tain
ad
iffe
ren
tial
dia
gn
osi
s.T
he
dis
crim
inat
ive
anal
ysi
so
fth
ese
spec
tra
has
dem
on
stra
ted
that
ph
eny
lala
nin
e,D
NA
,an
dam
ide
Isp
ectr
alv
aria
ble
sst
oo
do
ut
inth
ed
iffe
ren
tiat
ion
of
the
thre
eg
rou
ps.
Th
ep
erce
nta
ge
of
corr
ectl
ycl
assi
fied
gro
up
sb
ased
on
thes
eth
ree
bio
chem
ical
con
stit
uen
tsw
as9
3.1
%.
Th
is
resu
ltd
emo
nst
rate
dth
atF
T-R
aman
spec
tro
sco
py
isca
pab
leo
fd
iffe
ren
tiat
ing
mel
ano
ma
fro
m
its
met
asta
sis,
asw
ell
asfr
om
no
rmal
skin
Oli
vei
raet
al.
(20
10
)
Mel
ano
ma
Bas
alce
llca
rcin
om
a
Nea
r-in
frar
edR
aman
spec
tro
met
er
wit
hR
aman
pro
be
k=
83
0n
mP
=5
0–
20
0m
W
t exp
=2
0s
exp
osu
reti
me
Ato
tal
14
5R
aman
spec
tra
fro
mb
iop
syfr
agm
ents
of
no
rmal
,B
CC
,an
dm
elan
om
aw
ere
anal
yze
dto
iden
tify
dif
fere
nce
sin
the
bio
chem
ical
con
stit
uti
on
of
thes
esa
mp
les.
Ram
an
spec
tra
of
som
eco
mp
ou
nd
sw
hic
har
eex
pec
ted
tob
ere
pre
sen
ted
inh
um
ansk
insp
ectr
aw
ere
coll
ecte
dan
da
lin
ear
leas
t-sq
uar
esfi
ttin
gm
od
elto
esti
mat
eth
eco
ntr
ibu
tio
ns
of
thes
e
com
po
un
ds
toth
eti
ssu
esp
ectr
aw
asd
evel
op
ed.
Th
ere
sult
sh
ave
sho
wn
that
acti
n,
coll
agen
,
elas
tin
,an
dtr
iole
inw
ere
the
mo
stim
po
rtan
tb
ioch
emic
als
rep
rese
nti
ng
the
spec
tral
feat
ure
so
f
skin
tiss
ues
.A
clas
sifi
cati
on
mo
del
app
lied
toth
ere
lati
ve
con
trib
uti
on
of
coll
agen
III,
elas
tin
,
and
mel
anin
usi
ng
Eu
clid
ean
dis
tan
ceas
ad
iscr
imin
ato
rco
uld
dif
fere
nti
ate
no
rmal
fro
mB
CC
and
mel
ano
ma
Sil
vei
raet
al.
(20
12
)
Bas
alce
llca
rcin
om
a–
Th
ep
ote
nti
alu
seo
fR
aman
mic
rosp
ectr
osc
op
yfo
rau
tom
ated
eval
uat
ion
of
exci
sed
skin
tiss
ue
du
rin
gM
oh
sm
icro
gra
ph
icsu
rger
yw
asin
ves
tig
ated
.A
mu
ltiv
aria
tesu
per
vis
edcl
assi
fica
tio
n
mo
del
was
dev
elo
ped
and
val
idat
edo
n3
29
Ram
ansp
ectr
ao
fsk
inti
ssu
eac
qu
ired
fro
m2
0
pat
ien
ts.
Th
ere
sult
sh
ave
sho
wn
that
BC
Cca
nb
ed
iscr
imin
ated
fro
mh
ealt
hy
tiss
ue
wit
h
90
±9
%se
nsi
tiv
ity
and
85
±9
%sp
ecifi
city
.W
hen
this
mo
del
was
app
lied
on
tiss
ue
sect
ion
sfr
om
new
pat
ien
ts,
the
Ram
anim
ages
hav
esh
ow
nan
exce
llen
tco
rrel
atio
nw
ith
his
top
ath
olo
gic
alse
ctio
ns,
BC
Cb
ein
gd
etec
ted
inal
lp
osi
tiv
ese
ctio
ns
Lar
rao
na-
Pu
yet
al.
(20
09
)
Bas
alce
llca
rcin
om
aF
T-R
aman
spec
tro
met
er(R
FS
10
0/S
-Bru
ker
Inc.
,K
arls
ruh
e,
Ger
man
y)
Ex
cita
tio
nso
urc
e:N
d:Y
AG
lase
r
k=
1,0
64
nm
P=
30
0m
W
reso
luti
on*
4cm
-1
Eig
ht
set
of
sam
ple
sh
isto
pat
ho
log
ical
lyd
iag
no
sed
asB
CC
and
fiv
ese
to
fsa
mp
les
dia
gn
ose
das
ben
ign
tiss
ue
wer
ean
aly
zed
by
FT
-Ram
ansp
ectr
osc
op
yin
ord
erto
det
ect
spec
tral
chan
ges
bet
wee
nb
enig
nan
dm
alig
nan
tsk
inti
ssu
es.
As
are
sult
of
this
stu
dy
,th
eau
tho
rsd
emo
nst
rate
d
that
by
app
lyin
gp
rin
cip
alco
mp
on
ents
anal
ysi
so
ver
all
13
sam
ple
s,ti
ssu
ety
pe
cou
ldb
e
iden
tifi
edw
ith
sen
siti
vit
yan
dsp
ecifi
city
of
10
0%
Nu
nes
etal
.(2
00
3)
1096 J Cancer Res Clin Oncol (2013) 139:1083–1104
123
analysis of the Raman spectra was performed with various
methods, from partial least-squares regression (Zhao et al.
2008; Lui et al. 2012; Silveira et al. 2012), discriminative
analysis (Larraona-Puy et al. 2009; Oliveira et al. 2010;
Cartaxo et al. 2010; Lui et al. 2012), comparison of relevant
spectral bands (Nunes et al. 2003) to more complex
approaches like neural network analysis (Gniadecka et al.
2004). Most of the articles relied on more than one analyzing
technique in order to obtain reliable results, demonstrating
that a complex analysis is far superior to limited ones,
because changes in the spectra are rarely punctual.
Basal cell carcinomas were addressed in many studies
(Gniadecka et al. 2004; Lieber et al. 2008; Larraona-Puy
et al. 2009; Zeng et al. 2011; Lui et al. 2012; Silveira et al.
2012). In vivo diagnostic approaches showed great sensi-
tivities (all above 90 %), with lesser specificity (ranging
from 54 to 95 %). These results prove this method to be a
valuable adjunct to clinical examination in planning the
surgical excision. Sample studies (Nunes et al. 2003;
Gniadecka et al. 2004; Larraona-Puy et al. 2009; Silveira
et al. 2012) gave equally good results with sensitivities
near to 100 % and specificities above 85 % (Nunes et al.
2003; Larraona-Puy et al. 2009). Therefore, RS is soon
expected to compete with histological examination during
surgery, being faster and easier to perform, even in a future
adaptation of Moh’s micrographic surgery (Larraona-Puy
et al. 2009). In terms of single-band discrimination, amid I
proteins (around 1,660 cm-1) proved to be an important
element in differentiation between normal skin or mela-
noma (Nunes et al. 2003; Larraona-Puy et al. 2009),
although a more complex analysis seems to be more reli-
able (Gniadecka et al. 2004; Silveira et al. 2012). In regard
to differential diagnosis between BCC and SCC, the reports
are not conclusive, this direction of investigation being one
of the great importance and needing further research, as
long as excision planning is generally based on preopera-
tive data concerning this matter.
Differentiating benign pigmented lesions from melanoma
is a matter of great concern for the surgeon, being known that
this is the most aggressive skin cancer. Therefore, many
studies addressed this subject (Gniadecka et al. 2004; Zhao
et al. 2008; Cartaxo et al. 2010; Oliveira et al. 2010; Zeng
et al. 2011; Lui et al. 2012; Silveira et al. 2012). In vivo
examination (Zhao et al. 2008; Zeng et al. 2011; Lui et al.
2012) is of the upmost importance, as it directs the surgical
therapy. The results were outstanding, showing sensitivities
of nearly 100 % and specificities ranging from 70 to 78 %
(Zhao et al. 2008; Zeng et al. 2011). As dermoscopy is in
current use in preoperative evaluation of pigmented lesions,
research comparing it to RS is mandatory before bringing the
latter into standard medical practice.
The sample studies were even more encouraging,
showing that RS is well suited to distinguish melanoma
from other pigmented lesions (Gniadecka et al. 2004;
Cartaxo et al. 2010), from normal skin or metastatic tumors
(Oliveira et al. 2010; Silveira et al. 2012) or BCCs (Gni-
adecka et al. 2004; Silveira et al. 2012). Spectral bands that
can differentiate melanoma from normal skin, pigmented
nevi, or BCC include lipids (Gniadecka et al. 2004),
polysaccharides, tyrosine and amide I (Cartaxo et al. 2010),
collagen III, elastin and melanin (Silveira et al. 2012),
phenylalanine, DNA, and amide I (Oliveira et al. 2010),
showing a great deal of variability among articles, a fact
that points out the need for complex analysis of the spectra
in order to generate reliable results.
Two of the most important advantages of this method
are its applicability both in vivo and in vitro as well as its
rapidity in examination and results. Together with the good
sensibility and specificity figures, these make RS one of the
most valuable optical diagnostic methods in differentiating
benign, premalignant, and malignant skin lesions with a
good chance to enter common medical practice.
Confocal microscopy
Confocal microscopy (CM) is an optical imaging technique
for noninvasive tissue imaging with a higher resolution and
contrast than the conventional microscopy. These main
features derive from the usage of a point source of light for
the illumination of specimen and the placement of a pin-
hole between tissue specimen and detector which rejects
multiply scattered out-of-focus light allowing only the in-
focus light from the specimen to reach the detector. The
image created in this way is an optical section representing
one focal plane within the examined specimen. By
changing the plane of focus or moving the specimen, a
series of images at different positions can be produced
through the thickness of the specimen and a three-dimen-
sional representation of the specimen can be produced by
the optical sectioning.
This technique has become a valuable tool in derma-
tology during the last decade being applied in both basic
research (Rigby and Goldie 1999; Lima et al. 2009) and
clinical diagnosis including the assessment of benign and
malignant skin lesions (Gerger et al. 2006; Gonzalez 2009;
Ulrich et al. 2008; 2012a, b), tumor margin mapping
(Curiel-Lewandrowski et al. 2004; Scope et al. 2010),
monitoring response to medical treatments (Ulrich et al.
2010, 2012a, b), and diagnosis of inflammatory and
infective skin diseases (Hicks et al. 2003; Swindells et al.
2004). The performance of CM applications in the skin
cancer diagnosis as reported in the literature over the past
decade is presented in Table 5.
We can see from Table 5 that the great majority of the
cited studies have used devices from the same producer
(Vivascope 1000; Lucid Inc., Rochester, NY, USA or
J Cancer Res Clin Oncol (2013) 139:1083–1104 1097
123
Ta
ble
5C
lin
ical
app
lica
tio
ns
of
con
foca
lm
icro
sco
py
tech
niq
ue
(CM
)fo
rth
ed
iag
no
sis
of
skin
can
cer
Dis
ease
Dev
ice/
par
amet
ers
Res
ult
sR
efer
ence
Mel
ano
cyti
csk
in
lesi
on
s
ben
ign
nev
i
mal
ign
ant
mel
ano
mas
bas
alce
llca
rcin
om
as
seb
orr
hei
ck
erat
ose
s
NIR
refl
ecta
nce
con
foca
lm
icro
sco
pe
(Viv
asco
pe
10
00
;L
uci
dIn
c.,
Ro
ches
ter,
NY
,U
SA
).
lig
ht
sou
rce:
dio
de
lase
r
k=
83
0n
m;
P\
35
mW
nu
mer
ical
aper
ture
:0
.9
late
ral
reso
luti
on
:(0
.5–
1.0
)lm
axia
lre
solu
tio
n:
(3.0
–5
.0)
lm
Inth
isst
ud
y,
11
7m
elan
ocy
tic
skin
lesi
on
san
d4
5n
on
-mel
ano
cyti
csk
inle
sio
ns
(90
ben
ign
nev
i,2
7m
alig
nan
tm
elan
om
as,
15
bas
alce
llca
rcin
om
as,
and
30
seb
orr
hei
ck
erat
ose
s)
wer
eex
amin
edb
yC
M.
Th
eac
qu
ired
imag
esw
ere
rate
db
y4
ind
epen
den
to
bse
rver
s.T
he
resu
lts
of
the
eval
uat
ion
hav
esh
ow
nth
at:
the
dif
fere
nti
atio
nb
etw
een
mel
ano
ma
and
all
oth
erle
sio
ns
can
be
ach
iev
edw
ith
ap
osi
tiv
e
pre
dic
tiv
ev
alu
eo
f9
4.2
2%
;
mal
ign
ant
lesi
on
s(m
elan
om
aan
db
asal
cell
carc
ino
ma)
can
be
dia
gn
ose
dw
ith
ap
osi
tiv
e
pre
dic
tiv
ev
alu
eo
f9
6.3
4%
;
asse
ssm
ent
of
dis
tin
ctco
nfo
cal
mic
rosc
op
yfe
atu
res
sho
wed
ast
ron
gin
tero
bse
rver
corr
elat
ion
;
clas
sifi
cati
on
and
reg
ress
ion
tree
anal
ysi
sh
asfa
cili
tate
da
corr
ect
clas
sifi
cati
on
in9
6.3
0%
of
mel
ano
mas
,9
8.8
9%
of
ben
ign
nev
i,an
d1
00
%o
fb
asal
cell
carc
ino
mas
and
seb
orr
hei
c
ker
ato
ses.
Th
ese
resu
lts
dem
on
stra
teth
atC
Mm
ayb
eco
nsi
der
edas
ap
rom
isin
gm
eth
od
for
the
no
nin
vas
ive
asse
ssm
ent
of
mel
ano
ma
and
no
n-m
elan
om
ask
intu
mo
rs
Ger
ger
etal
.(2
00
6)
Mal
ign
ant
mel
ano
mas
ben
ign
nae
vi
NIR
refl
ecta
nce
con
foca
lm
icro
sco
pe
(Viv
asco
pe
10
00
;L
uci
dIn
c.,
Ro
ches
ter,
NY
,U
SA
)
Ato
tal
of
3,7
09
tum
or
imag
eso
bta
ined
fro
m2
0m
alig
nan
tm
elan
om
asan
d5
0b
enig
nn
aev
i
wer
eev
alu
ated
by
ind
epen
den
to
bse
rver
s.T
he
resu
lts
hav
esh
ow
nth
atse
nsi
tiv
ity
and
spec
ifici
tyo
f9
7.5
and
99
%co
uld
be
ach
iev
edb
yth
ein
dep
end
ent
ob
serv
ers
(po
siti
ve
pre
dic
tiv
ev
alu
e9
7.5
%,
neg
ativ
ep
red
icti
ve
val
ue
99
%).
Cla
ssifi
cati
on
tree
anal
ysi
sh
as
con
du
cted
toa
corr
ect
clas
sifi
cati
on
in9
2.4
%o
fth
eb
enig
nn
evu
sim
ages
and
97
.6%
of
mel
ano
ma
imag
es.
Co
nse
qu
entl
y,
CM
cou
ldb
eu
sed
asa
scre
enin
gto
ol
insk
ino
nco
log
y
Ger
ger
etal
.(2
00
8)
Mel
ano
ma
nev
i
Inth
isst
ud
y,
the
sen
siti
vit
yan
dsp
ecifi
city
of
con
foca
lfe
atu
res
for
the
dia
gn
osi
so
f
equ
ivo
cal
mel
ano
cyti
cle
sio
ns
(13
6m
elan
om
asan
d2
15
nev
i)w
ere
eval
uat
ed.
Mic
rosc
op
y
imag
ean
aly
sis
by
v2te
st,m
ult
ivar
iate
dis
crim
inan
tan
aly
sis,
and
bin
ary
log
isti
cre
gre
ssio
n
rev
eale
dth
at:
mel
ano
mas
are
mo
stly
char
acte
rize
db
yep
ider
mal
dis
arra
yan
dp
aget
oid
cell
sin
the
epid
erm
is,
no
n-e
dg
edp
apil
lae,
and
cell
ula
rat
yp
iaat
the
jun
ctio
n,
and
aty
pic
aln
ests
and
bri
gh
tn
ucl
eate
dce
lls
inth
eu
pp
erd
erm
is;
ben
ign
lesi
on
sar
ech
arac
teri
zed
by
reg
ula
rd
erm
al–
epid
erm
alar
chit
ectu
re,
and
abse
nce
of
pag
eto
idin
filt
rati
on
and
aty
pic
alce
lls;
fiv
eo
ut
of
the
13
6m
elan
om
as,
wit
hm
ild
lyat
yp
ical
mel
ano
cyte
san
do
ccas
ion
alp
aget
oid
cell
sat
his
top
ath
olo
gy
,w
ere
no
td
iag
no
sed
by
con
foca
lm
icro
sco
py
.T
hes
ere
sult
sle
adto
the
con
clu
sio
nth
atC
Mco
uld
be
use
ful
for
seco
nd
lev
elex
amin
atio
no
fcl
inic
aleq
uiv
oca
l
lesi
on
s
Pel
laca
ni
etal
.(2
00
7)
Mel
ano
ma
nev
i
Sp
itz
or
Ree
dn
evi
NIR
refl
ecta
nce
con
foca
lla
ser
scan
nin
g
mic
rosc
op
es(V
ivas
cop
e1
00
0an
d
Viv
asco
pe
15
00
;L
uci
dIn
c.,
Ro
ches
ter,
NY
,U
SA
)
Ato
tal
of
20
2m
elan
ocy
tic
lesi
on
s(7
6m
elan
om
as,
11
4n
evi,
and
12
Sp
itz
or
Ree
dn
evi)
wer
ein
ves
tig
ated
inth
isst
ud
yin
ord
erto
esta
bli
sha
corr
elat
ion
bet
wee
nd
erm
osc
op
ic
pat
tern
sin
mel
ano
cyti
cle
sio
ns
and
con
foca
lm
icro
sco
pic
fin
din
gs
and
con
ven
tio
nal
his
top
ath
olo
gic
fin
din
gs.
So
me
char
acte
rist
icar
chit
ectu
ral
and
cyto
log
icsu
bst
rate
sw
ere
iden
tifi
edb
yco
nfo
cal
mic
rosc
op
yan
dco
rrel
ated
wit
hh
isto
pat
ho
log
ical
exam
inat
ion
.T
he
resu
lts
of
this
stu
dy
cou
ldb
eu
sefu
lfo
rth
eid
enti
fica
tio
no
fsp
ecifi
csu
bst
rate
sin
mel
ano
cyti
cle
sio
ns
and
con
seq
uen
tly
the
inte
rpre
tati
on
of
the
der
mo
sco
pic
feat
ure
s
Pel
laca
ni
etal
.(2
00
8)
1098 J Cancer Res Clin Oncol (2013) 139:1083–1104
123
Ta
ble
5co
nti
nu
ed
Dis
ease
Dev
ice/
par
amet
ers
Res
ult
sR
efer
ence
Mel
ano
ma
mel
ano
cyti
cn
evi
Ato
tal
of
12
5p
atie
nts
wit
h1
25
lesi
on
s(8
8m
elan
ocy
tic
nev
ian
d3
7m
elan
om
as)
wer
e
exam
ined
by
CM
and
der
mo
sco
py
.T
he
resu
lts
of
this
stu
dy
rev
eale
dth
atth
ed
erm
osc
op
y
had
ase
nsi
tiv
ity
of
89
.2%
,a
spec
ifici
tyo
f8
4.1
%,
ap
osi
tiv
ep
red
icti
ve
val
ue
of
70
.2%
,
and
an
egat
ive
pre
dic
tiv
ev
alu
eo
f9
4.9
%w
hil
eth
eC
Mw
asfo
un
dto
hav
ea
sen
siti
vit
yo
f
97
.3%
,a
spec
ifici
tyo
f8
3.0
%,
ap
osi
tiv
ep
red
icti
ve
val
ue
of
70
.6%
,an
da
neg
ativ
e
pre
dic
tiv
ev
alu
eo
f9
8.6
%.
Th
ese
resu
lts
sug
ges
tth
atth
ese
two
dia
gn
ost
icm
eth
od
sar
e
com
ple
men
tary
Lan
gle
yet
al.
(20
07
)
Bas
alce
llca
rcin
om
a
squ
amo
us
cell
carc
ino
ma
NIR
refl
ecta
nce
con
foca
lm
icro
sco
pe
(Viv
asco
pe
10
00
;L
uci
dIn
c.,
Ro
ches
ter,
NY
,U
SA
)
Inth
isst
ud
y,
the
CM
and
his
top
ath
olo
gy
wer
eu
sed
asm
eth
od
sfo
rth
eex
amin
atio
no
fth
e
dif
ficu
lt-t
o-d
iag
no
sesk
inle
sio
ns
in1
37
pat
ien
ts.
Th
ere
sult
sh
ave
pro
ved
that
10
6le
sio
ns
(ou
to
fa
tota
lo
f1
29
lesi
on
sh
isto
log
ical
lyp
rov
ento
be
mal
ign
anci
es)
wer
ed
iag
no
sed
as
‘‘m
alig
nan
t’’
by
CM
.A
dif
fere
nce
of
23
lesi
on
sw
ere
dia
gn
ose
das
‘‘n
orm
al’’
by
CM
(6
bas
alce
llca
rcin
om
aan
d1
7sq
uam
ou
sce
llca
rcin
om
a)d
emo
nst
rati
ng
afa
lse
neg
ativ
era
te
of
23
/12
9(1
7.8
3%
)o
ra
sen
siti
vit
yo
f8
2.1
7%
.T
hes
ere
sult
ssh
ow
that
CM
can
pro
vid
e
dia
gn
ost
icin
form
atio
nw
hic
his
reli
able
for
ov
er8
2%
of
clin
ical
lyd
iffi
cult
tod
iag
no
se
Am
jad
iet
al.
(20
11
)
Bas
alce
llca
rcin
om
a
acti
nic
ker
ato
sis
Flu
ore
scen
cem
icro
sco
pe
(Op
tiS
can
Ltd
.,
Mel
bo
urn
e,A
ust
rali
a)
Inth
isst
ud
y,
flu
ore
scen
tco
nfo
cal
mic
rosc
op
yw
asu
sed
for
the
mo
nit
ori
ng
of
acti
nic
ker
ato
ses
and
bas
alce
llca
rcin
om
are
spo
nse
toto
pic
alth
erap
yu
sin
gIm
iqu
imo
das
an
imm
un
e-re
spo
nse
mo
difi
er.
Th
ere
sult
sd
emo
nst
rate
dth
atth
iso
pti
cal
tech
niq
ue
allo
wed
a
mo
nit
ori
ng
of
the
loca
lim
mu
ne
resp
on
sefo
llo
win
gth
erap
yw
ith
Imiq
uim
od
and
dem
on
stra
ted
aco
nti
nu
ou
sn
orm
aliz
atio
no
fd
isea
sed
skin
on
rep
eate
dev
alu
atio
ns
ov
er
tim
e.T
hes
ere
sult
sar
eo
nly
pre
lim
inar
yan
dfu
rth
erin
ves
tig
atio
ns
are
req
uir
edin
the
futu
re
Ast
ner
etal
.(2
00
8)
Act
inic
ker
ato
sis
NIR
refl
ecta
nce
con
foca
lm
icro
sco
pe
(Viv
asco
pe
10
00
;L
uci
dIn
c.,
Ro
ches
ter,
NY
,U
SA
)
Fo
rty
-six
AK
sfr
om
44
pat
ien
tsw
ere
incl
ud
edin
this
stu
dy
.T
he
eval
uat
ion
of
thes
ele
sio
ns
was
per
form
edb
ycl
inic
alex
amin
atio
n,
CM
,an
dro
uti
ne
his
tolo
gy
.T
he
resu
lts
of
this
stu
dy
hav
esh
ow
nth
atth
ese
nsi
tiv
ity
/sp
ecifi
city
val
ues
of
CM
feat
ure
sra
ng
edfr
om
80
to
98
.6%
.T
hes
ere
sult
ssh
ow
that
CM
cou
ldb
eco
nsi
der
edas
anad
jun
ctto
ol
tocl
inic
al
dia
gn
osi
san
dm
on
ito
rin
g
Ulr
ich
etal
.(2
00
8)
Len
tig
om
alig
na
sola
rle
nti
go
eph
elis
acti
nic
ker
ato
sis
flat
seb
orr
hei
ck
erat
osi
s
NIR
refl
ecta
nce
con
foca
lm
icro
sco
pe
(Viv
asco
pe
10
00
;L
uci
dIn
c.,
Ro
ches
ter,
NY
,U
SA
)
Inth
isst
ud
y,
ato
tal
of
64
CM
feat
ure
sw
ere
sco
red
retr
osp
ecti
vel
yan
db
lin
ded
tod
iag
no
sis
ina
seri
eso
fC
Msa
mp
led
,cl
inic
ally
equ
ivo
cal,
mac
ule
so
fth
efa
ce(n
=8
1le
nti
go
mal
ign
a,n
=2
03
ben
ign
mac
ule
s).
Inad
dit
ion
tod
escr
ibin
gC
Md
iag
no
stic
feat
ure
sfo
r
len
tig
om
alig
na,
anal
go
rith
mw
asd
evel
op
ed(L
Msc
ore
)to
dis
tin
gu
ish
len
tig
om
alig
na
fro
mb
enig
nm
acu
les.
Th
ere
sult
sh
ave
sho
wn
that
aL
Msc
ore
of
X2
resu
lted
ina
sen
siti
vit
yo
f8
5%
and
spec
ifici
tyo
f7
6%
for
the
dia
gn
osi
so
fle
nti
go
mal
ign
a
Gu
iter
aet
al.
(20
10
)
Len
tig
ines
len
tig
om
alig
na
Ten
pat
ien
tssu
spec
ted
wit
hle
nti
gin
es(n
=6
)an
dle
nti
go
mal
ign
as(n
=4
)w
ere
incl
ud
ed
inth
isst
ud
y.
Th
ech
arac
teri
stic
so
fth
ese
pig
men
ted
lesi
on
sw
ere
qu
alit
ativ
ely
des
crib
ed
by
CM
and
com
par
edw
ith
his
top
ath
olo
gic
fin
din
gs.
Th
ere
sult
ssh
ow
edth
efo
llo
win
g:
the
ben
ign
len
tig
ines
pre
sen
ted
dis
tin
ctar
chit
ectu
ral
and
cyto
log
icfe
atu
res
com
par
edw
ith
mel
ano
mas
;
anin
crea
sein
the
den
sity
of
der
mal
pap
illa
esu
rro
un
ded
by
ab
rig
ht
mo
no
mo
rph
icla
yer
of
cell
sw
asse
enin
all
case
so
fle
nti
gin
es,
bu
tn
ot
for
mel
ano
mas
;
len
tig
ines
pre
sen
ted
anab
sen
ceo
fat
yp
ical
mel
ano
cyte
s;m
elan
om
asp
rese
nte
db
rig
ht,
aty
pic
al,p
oly
mo
rph
ou
sce
lls
pre
sen
tin
ap
aget
oid
pat
tern
wit
hco
arse
,b
ran
chin
gd
end
rite
s
ob
serv
edth
rou
gh
ou
tth
eep
ider
mis
.T
hes
ech
arac
teri
stic
so
fle
nti
gin
esco
uld
hel
pin
the
dia
gn
osi
so
fm
elan
om
aan
dd
iscr
imin
atio
nfr
om
ben
ign
lesi
on
s
Lan
gle
yet
al.
(20
06
)
J Cancer Res Clin Oncol (2013) 139:1083–1104 1099
123
Vivascope 1500, Lucid-Tech Inc., Henrietta, NY, USA),
the only problem that the researchers had to answer being
the criteria in image analysis is that it can give the most
accurate result. Only one article (Astner et al. 2008) used a
fluorescence microscope that allowed also the evaluation of
the effectiveness of the treatment with Imiquimod for the
selected lesions.
Differentiating in a safe and reliable mode benign pig-
mented lesions from melanoma remains the main concern
of the researchers (Gerger et al. 2006, 2008; Langley et al.
2006, 2007; Pellacani et al. 2007, 2008; Ahlgrimm-Siess
et al. 2009; Guitera et al. 2010). While the technique seems
to be the same in most studies, it is the interpretation that
makes the difference. While some authors used assessment
from more than one investigator (Gerger et al. 2006, 2008;
Pellacani et al. 2007), others relied on only one expert
(Langley et al. 2006, 2007; Pellacani et al. 2008; Ahl-
grimm-Siess et al. 2009; Guitera et al. 2010). Features that
lead to a melanoma or lentigo maligna diagnostic include:
epidermal disarray and pagetoid cells in the epidermis,
non-edged papillae and disarrangement of the dermoepi-
dermal junction, cellular atypia, atypical nests, and bright
nucleated cells in the dermis or papillae (Pellacani et al.
2007, 2008; Guitera et al. 2010) and so on. Most
researchers have tried to find a reliable interpretation
algorithm that may be applicable to all cases (Gerger et al.
2006, 2008; Pellacani et al. 2007; Guitera et al. 2010) or at
least point out the main significant aspects (Langley et al.
2006; Pellacani et al. 2008; Ahlgrimm-Siess et al. 2009).
Confocal microscopy proved to be a valuable adjunct to
dermoscopy (Langley et al. 2007; Pellacani et al. 2008).
Sensitivity and specificity indices were high (above 80 %)
(Gerger et al. 2006, 2008; Pellacani et al. 2007; Guitera
et al. 2010), proving that the method may become a useful
tool in preoperative decisions. It has the advantage of being
quick and it does not need complex mathematical inter-
pretation (like other cited optical methods do), but it still
relies on expert opinion, as long as a proven algorithm is
difficult to establish even with meta-analysis.
Less attention was given to non-melanoma skin cancers
(Astner et al. 2008; Ulrich et al. 2008; Amjadi et al. 2011),
but the results proved CM to be a good pretreatment
assessment tool, both in diagnostic (Amjadi et al. 2011) or
even in orienting the surgical excision margins (Amjadi
et al. 2011). A derivative method that uses fluorescence has
shown its value in monitoring noninvasive therapies on this
type of skin malignancies (Astner et al. 2008).
Discussion
Optical techniques (OCT, DFT, DRS, RS, and CM) have
shown promising results in the diagnosis of skin cancer.Ta
ble
5co
nti
nu
ed
Dis
ease
Dev
ice/
par
amet
ers
Res
ult
sR
efer
ence
Len
tig
om
alig
na
len
tig
om
alig
na
mel
ano
ma
Tw
elv
ep
atie
nts
wit
h1
7le
sio
ns
(len
tig
om
alig
na
and
len
tig
om
alig
na
mel
ano
ma)
wer
e
exam
ined
by
con
foca
lm
icro
sco
py
and
his
tolo
gic
alex
amin
atio
n.
Th
ere
sult
so
fth
isst
ud
y
sho
wed
that
mai
nfe
atu
res
of
thes
ele
sio
ns
ob
serv
edin
the
mic
rosc
op
icim
ages
are
rela
ted
toa
foca
lin
crea
sein
aty
pic
alm
elan
ocy
tes
and
nes
tssu
rro
un
din
gad
nex
alo
pen
ing
s,sh
eets
of
mai
nly
den
dri
tic
mel
ano
cyte
s,co
rd-l
ike
rete
rid
ges
atth
ed
erm
oep
ider
mal
jun
ctio
nan
d
anin
filt
rati
on
of
adn
exal
stru
ctu
res
by
aty
pic
alm
elan
ocy
tes.
Th
ese
resu
lts
sug
ges
tth
atth
e
inv
ivo
asse
ssm
ent
of
equ
ivo
cal
skin
lesi
on
sat
ace
llu
lar
lev
elco
uld
be
do
ne
by
CM
Ah
lgri
mm
-Sie
sset
al.
(20
09
)
1100 J Cancer Res Clin Oncol (2013) 139:1083–1104
123
They do not usually require tissue removal and allow the
real-time diagnosis, but some of them (RS, CM) can also
be applied on tissue samples, being more rapid than path-
ological examination.
Optical coherence tomography proved to be an effective
imaging technique for the investigation of skin morphology
alongside with fluorescence and DRS. The last two spec-
troscopic techniques have been widely applied to acquire
information not only about the structure of tissue but also
about biochemical composition of tissue. Raman spec-
troscopy has the same goal of biochemical characterization
of tissues, but has shown better results in establishing
differential diagnosis between benign, premalignant, and
malignant skin lesions. Confocal spectroscopy allows for a
more direct visual evaluation of skin tumors, being closest
to pathological examination and does not rely on compli-
cated analyzing models.
In OCT, diagnostic accuracy in clinical diagnosis of AK
ranges from 79 to 86 % in sensitivity and 83 to 100 % in
specificity. Several reports have demonstrated for BCC and
AK an excellent correlation between fluorescence pattern
and histopathology. The fluorescence spectroscopy could
be therefore used in clinically ill-defined (malignant, pre-
malignant, or benign) cutaneous lesions in order to better
delimitate neoplastic tissue. Diffuse reflectance spectros-
copy has the potential to provide the means to identify
precancerous and cancerous lesion. Tissue information
obtained by this spectroscopic technique could be useful in
tissue classification and in the detection and characteriza-
tion of a large number of pathologic disorders including
cancer. Raman spectroscopy showed even better results
than other cited spectroscopic techniques, its sensitivities
rising high above 90 % both for differential diagnosis
between benign and malignant tumors or even assessing
tumoral margins. Confocal microscopy has the advantage
of providing direct images of tumoral tissue and it does not
need complex mathematical interpretation; nevertheless, it
needs expert interpretation and is probably better accepted
by the physicians, because peers are directly involved in its
applications.
Widespread clinical application of these optical methods
in the diagnosis of skin cancer is conditioned by certain
factors such as the cost of equipments and their mainte-
nance, personnel training for the acquisition, processing
and interpretation of the data, and time of investigation.
The cost of equipments for these optical methods is dif-
ferent. The portable diffuse reflectance spectrometers are
relatively cheap while the fluorescence spectrometers
require a large investment because of the special light
source (laser or lamp) necessary to generate fluorescence.
The complexity and requirements for OCT systems con-
cerning the parameters of excitation light source and of the
detection module make the costs of the equipments to be
quite high. All these equipments require minimal mainte-
nance, especially related to their periodic calibration.
Operating all these equipments is not difficult and the staff
training is quite easy. The application of algorithms for
data analysis and the interpretation of results however
require highly qualified personnel and special attention
should be paid for their adequate training. Unlike other
techniques for the diagnosis of skin cancer that are time-
consuming (e.g., histopathological test), all these optical
diagnostic techniques allow for real-time diagnosis.
Conclusions
In conclusion, the noninvasive optical techniques presented in
this study (OCT, DFT, DRS, RS, CM) proved to be effective
in the diagnosis of both benign and malignant diseases of the
skin. Based on the presented results, we can anticipate that
these techniques will find their place in medical practice as
well as new advanced equipment for optical diagnostics will
be developed and released on the market. To achieve this, the
efforts of the scientific community, the medical community,
and manufacturers should converge.
Acknowledgments This work was financed by the Ministry of
Education, Research, Youth and Sport by means of the Research
Program no. PN II PCCA 184/2012.
Conflict of interest We declare that we have no conflict of interest.
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